bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Characterization of Human Immunity-Related GTPase Family M (IRGM)
Authors: Sabboobeh Sarah , Nagar Bhushan
Published: November 2015

The Immunity-Related GTPase Family M protein (IRGM) is involved in regulating cellular autophagy. Cellular knockdown of IRGM was shown to allow RNA viruses to hijack the autophagic immune response. Additionally, recent genetic studies have shown that underexpression of IRGM is associated with the incidence of Crohn's disease and infection by Mycobacterium tuberculosis. IRGM is an interferon-induced GTPase with an evolutionary conserved P-loop. It is an effector of the interferon-gamma pathway, but, unlike its protein family members, is not directly activated by the pathway. Its mechanism of action has been proposed to occur by translocation of IRGM to the mitochondria through recognition of cardiolipin, and affecting mitochondrial fission to induce autophagy. This potential interaction with cardiolipin might indicate the presence of a unique GTPase recognition and activation fold within IRGM. Our goal is to determine the X-ray crystal structure of IRGM in an effort to understand its molecular role in normal and diseased states. Additionally, we seek to test its interaction with and mechanism of recognition to mitochondrial cardiolipin as well as other autophagy-inducing binding partners. Currently, we have managed to express human IRGM in bacterial cells and have purified it to homogeneity using affinity and size-exclusion chromatography. These findings will serve to elucidate the mechanism of action of IRGM. Crucially, we hope to gain an understanding of its contributing role to Crohn's disease and tuberculosis infection at the molecular level, potentially paving the way to structure-based drug design and therapeutic opportunities.
Crystal structure of Schistosoma mansoni HGPRT isoform 1
Authors: Romanello Larissa , de Souza Juliana , Bird Louise , Nettleship Joanne , Owens Raymond , Reddivari Yamini , Brandão-Neto José , Pereira Humberto
Published: November 2015

Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 207 million people worldwide [1], and does not have the purine "de novo" pathway, depending entirely on the purine salvage pathway to supply its demands on purines [2]. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in this pathway and the only validated enzyme target of the pathway [3]. HGPRT catalyzes the PRPP dependent conversion of hypoxanthine/guanine to inosine monophosphate or guanine to guanosine monophosphate. HGPRT1 gene was amplified, cloned, expressed and purified at the Oxford Protein Production Facility (OPPF-UK). Robotic crystallization trials were performed and SmHGPRT crystallized in several conditions of the Morpheus crystallization kit: A4, A8, A9, and C9. The crystals appear about a day and have about 30 μM in greatest dimension. About a hundred crystals were screened with x-rays on the macromolecular crystallography beamlines I02 and I24 at Diamond Light Source. 21 datasets was collected from 2.97 to 4.11Å resolution. A solution was obtained for HGPRT1 belongs space group P212121 in a dataset to 3.4Å resolution, with four monomer in the ASU. The structure was solved by the program Phaser using HGPRT human as a search model. The refinement is being carried out by program Phenix. The density map is acceptable for the resolution but a great manual work of interpretation is necessary for the refinement of this structure. The most important is the demonstration that it was possible to crystallize and collect data of SmHGPRT. A major effort will be undertaken to improve the size and diffraction power of HGPRT crystals as well as in the resolution of the structure of HGPRT in other space groups. This structure will increase the structural information available about the Schistosoma mansoni purine salvage pathway.
Structural insights into heme oxygenase-1 inhibition by azole-based compounds
Authors: Rahman Mona , Vukomanovic Dragic , Vlahakis Jason , Szarek Walter , Nakatsu Kanji , Jia Zongchao
Published: November 2015

The development of heme oxygenase (HO) inhibitors, especially those that are isozyme-selective, promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. HO is known to have cytoprotective properties with a role in several disease states; thus, it is an enticing therapeutic target. Traditionally, given their structural similarity with heme, the metalloporphyrins have been used as competitive HO inhibitors. However, given heme's important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), nonselectivity is an unfortunate side-effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort to develop novel compounds as potent, selective inhibitors of HO. This resulted in the creation of non-competitive HO-selective inhibitors, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated, providing insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. The structures revealed a common binding mode, despite different structural fragments, with the flexibility to accommodate bulkier substituents via "induced fit". Compounds bind to the distal side of heme through an azole ``anchor" which coordinates with the heme iron. Expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds, with a distal hydrophobic pocket providing further stabilization yet without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140.
Vanin-1- a human ectoenzyme involved in inflammation, metabolism and disease
Authors: Peat Thomas , Boersma Ykelien , Newman Janet , Adams Tim , Krippner Guy , Bozaoglu Kiymet
Published: November 2015

Inflammation is steadily gaining recognition as contributing to different disease states, e.g. cancer, immune dysfunction and cardiovascular disease. Vanin-1, a GPI-anchored, developmentally regulated member of the nitrilase family, sits at the intersection of many known pathways of inflammation. Three members of the vanin family of enzymes in humans have been described, with vanin-1 and vanin-2 having confirmed enzymatic activity. A known substrate is pantetheine which is hydrolyzed to give vitamin B5 and cysteamine. One function of these enzymes is in pantothenate recycling, and therefore they are involved in the Coenzyme A cycle and metabolism. Mouse knockout studies have shown that Vnn1(-/-) mice are resistant to oxidative stress, intestinal inflammation and colitis. Epidemiological studies have shown that mutations in vanin-1 in humans are associated with child obesity1; other studies show an association with cholesterol homeostasis and cardiovascular disease. Importantly, the metabolic pathways of lipid metabolism and inflammation are interconnected2 whereby impairment of lipid metabolism leads to inflammation and inflammation leads to impairment of lipid metabolism. We recently solved the structure of a soluble form of human vanin-1 using a single heavy atom derivative and anomalous scattering to 2.3 Å resolution. It has two domains: a predicted nitrilase domain and a cap domain which has no known sequence homology to any other structural domain. We also have structures with inhibitors bound and have performed mutational studies to determine the function of the cap domain and affirm the catalytic residues in the catalytic site. Structural studies have been complemented by enzymatic assays showing various levels of activity for the mutant and wild type enzymes. These data will be fundamental in characterizing vanin-1 in different disease states.
Toxins from TA system of Helicobacter pylori and insight into mRNase activity
Authors: Pathak Chinar , Im Hookang , Jang Sun-bok , Yang Yeon-Jin , Yoon Hye-Jin , Kim Hong-Man , Kwon Ae-Ran , Lee Bong-Jin
Published: November 2015

The toxin-antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. The HP0892-HP0893 and HP0894-HP0895 toxin-antitoxin systems are the only two known TA systems belonging to Helicobacter pylori. In both of these TA systems, the antitoxin binds and inhibits the toxin and regulates the transcription of the TA operon. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remains unclear. Therefore, here an attempt was made to shed some light on the mechanism behind the TA system of HP0892-HP0893 and HP0894-HP0895. Here, we present the crystal structures of apo- and copper-bound HP0894 at 1.28 Å and 1.89 Å, respectively, and the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues involving the mRNase active site was shown. Through the combined approach of structural analysis along with isothermal calorimetry studies and structural homology search, the amino acids involved in mRNase active site were monitored. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself acting as a general acid in an acid-base catalysis reaction, while His47 and His60 stabilize the transition state. Glu58 acts as a general base, and substrate reorientation is caused by Phe88. In the mRNase active site of HP0892 toxin, the most catalytically important residue, His86, reorients itself to exhibit RNase activity while Glu58 acts as a general base. His47 and His60 are considered to be involved in enzymatic activity. Glu58 and Asp64 are involved in substrate binding and specific sequence recognition. The mutational constructs were used for isothermal calorimetric studies to analyze the effect of catalytic residues.
Structural and functional studies of Sacsin
Authors: Menade Marie , Kozlov Guennadi , Gehring Kalle
Published: November 2015

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. It was first described in the French-Canadian population in 1978 by JP Bouchard, but ARSACS cases have since been found worldwide. The SACS gene codes for Sacsin, a 520kDa protein, which localizes in the cytoplasm, close to the mitochondria. Sacsin is composed of an N-terminal ubiquitin-like domain, which binds the proteasome, followed by three Sacsin repeat regions (SRR), a DnaJ domain, and at the C-terminus a nucleotide-binding HEPN domain, which mediates dimerization. While over 120 mutations in Sacsin are known to cause disease, the cellular function of the protein remains unclear. We recently crystallized the ATPase-like fragment of the first SRR domain from human Sacsin. The crystals diffract to better than 2A resolution and the structure determination is in progress. Future studies will focus on larger fragments of Sacsin as well as biochemical studies to investigate the cellular function of the protein.
Towards structural characterization of H. polymorpha telomerase components
Authors: Kallio Johanna , Rodina Elena , Zvereva Maria , Petrova Olga , Malyavko Alexandr , Smekalova Elena , Parfenova Julia , Hackenberg Claudia , Wiegels Tim , Dontsova Olga , Lamzin Victor
Published: November 2015

Telomeres are regions of non-coding DNA that cap the chromosomes, preventing the loss of coding DNA during cell division and contributing to chromosomal stability. In actively dividing cells, such as embryonic stem cells, the telomeres need to elongated by telomerase. The telomerase complex consist of the enzyme telomerase reverse transcriptase (TERT), telomerase RNA (TR) and additional proteins. TERT and TR are required for the telomerase activity in vitro. Telomerase is active in vast majority of the cancer cells ensuring continuous cell division and tumor growth. Syndromes leading to premature aging are often associated with short telomeres. Finding ways to regulate the telomerase activity would help to advance therapies for these conditions. However, the structural information available of the telomerase complex is very limited. We have chosen thermophilic yeast Hansenula polymorpha as a model system due to the stability of its proteins. The N-terminal domain of the TERT is essential for telomerase activity and possibly is involved in binding of TR, telomeric DNA and additional protein components of the telomerase complex. We have crystallised the N-terminal domain of H. polymorpha TERT and, in lack of a homologious structure, produced a seleno-methionine derivative of the protein. MAD data on N-terminal domain has been collected to resolution of 2.0 Å at the PETRA-III beamline P13 (EMBL/DESY) in Hamburg. We will discuss the structure-function relationship of the N-domain and the whole TERT component.
Substrate selectivity of C-terminal sucrase isomaltase and maltase glucoamylase
Authors: Jones Kyra , Rose David
Published: November 2015

Carbohydrates make up a significant component of the human diet. One approach to controlling blood glucose and serum insulin levels in individuals with type II diabetes is inhibition of intestinal α-glucosidases and pancreatic α-amylases. Two intestinal α-glucosidases, sucrase isomaltase (SI) and maltase glucoamylase (MGAM), are responsible for the final step of starch hydrolysis in mammals in the small intestine: the release of free glucose. Each enzyme consists of two catalytic subunits: N-terminal sucrase isomaltase (ntSI) and C-terminal sucrase isomaltose (ctSI); and N-terminal maltase glucoamylase (ntMGAM) and C-terminal maltase glucoamylase (ctMGAM). Here, residues hypothesized to impact substrate specificity of ctSI and ctMGAM will be presented, enhancing our understanding of the functionality of these enzymatic subunits as well as their overlapping substrate specificity.
Structural studies on 14-3-3ζ: Compounds that target the dimer interface
Authors: Dhagat Urmi , Woodcock Joanna , Tiong Chrystal , Holien Jessica , Coolen Carl , Broughton Sophie , Pitson Stuart , Hughes Tim , Lopez Angel , Parker Michael
Published: November 2015

14-3-3 proteins are a highly conserved family of dimeric phospho-serine binding proteins that modulate the functions of key cellular proteins involved in signaling. 14-3-3ζ plays a prominent role in signaling pathways leading to inhibition of apoptosis, sequestration of tumor suppressor proteins and activation of signalling pathways that promote growth. 14-3-3ζ expression is up-regulated in many human cancers and associated with enhanced survival of cancer cells. The significant association of 14-3-3ζ over expression with disease recurrence and chemo-resistance makes this protein an attractive candidate for anti-cancer therapy. The anti-apoptotic activity of 14-3-3ζ is entirely dependent on the dimeric state of the protein. Our studies have shown that 14-3-3ζ activity is regulated by sphingosine and other lipid analogs that render 14-3-3 phosphorylatable, disrupting its dimeric state thereby leading to apoptosis [1]. Structural studies and mutagenesis on 14-3-3ζ confirm that the dimeric state of 14-3-3ζ is stabilized by salt bridges that form across the dimer interface. Based on this we have carried out an in silico screen of a virtual library of drug-like small molecules to identify compounds that bind to the dimer interface of 14-3-3ζ. Candidate small molecules have been assessed for their ability to render 14-3-3ζ phosphorylatable in vitro and consequently we have identified a family of small molecules with 14-3-3ζ dimer-destabilizing properties. These small molecules induce apoptosis in leukemic cells by activating apoptotic mediators known to be regulated by dimeric 14-3-3. We have recently solved the crystal structure of 14-3-3ζ with one of our hit compounds bound at the dimer interface. Our results suggest that relatively small perturbations at the dimer interface, can destabilize the salt bridges that hold 14-3-3 dimers together, thus providing a novel approach to targeting 14-3-3 proteins for therapeutic benefit.
Type II cleaved gingipain adhesins act in bacterial invasion
Authors: Gao Jinlong , Vicari Daniele , Li Nan , Collyer Charles
Published: November 2015

Gingipains are multi domain peptidases that are primary virulence factors expressed by the keystone periodontal disease pathogen P. gingivalis. The adhesion regions of these proteases are multi-domain complexes that are comprised of a number of modules that belong to the type 1 (T1) family of gingipain adhesins (also known as cleaved adhesin domains). We have to date reported the crystal structures of three homologous variants of these 19 kDa T1 domains and shown that they recognise a number of host target proteins. We have proceeeded to predict from sequence analysis and binding data that in addition to the T1 domains the adhesion regions also contain a number of other structurally (and sequence) unrelated adhesins (referred to here as type 2 or T2 gingpain adhesins) that may synergistically contribute to the virulence of P. gingivalis. We have recombinantly expressed and crystallized the first example of an 17.5 kDA T2 adhesin coded for by a fragment of the gene for the lysine specific gingipain (kgp). We report here the structure refined at 1.05 angstroms resolution and thereby confirm our hypothesis of the existence of the T2 domain family. This structure represents a new fold family which is distantly similar in topology to that of the plastocyanin/azurin family of proteins but with no copper binding sites. Like the T1 adhesins it contains a structural binding site for calcium. We observe that the recombinant T2 adhesin found in the gingipain Kgp also binds directly to host target proteins but more importantly can also competitively inhibit a bacterial invasion cell based assay. The role of this new T2 adhesin in a specific adhesion activity in the cell invasion mechanism is likely a potential critical component in the overall virulence of this pathogen. Figure. Cartoon representation of the superposition of the structures of the Kgp T2 adhesin (green cartoon and blue calcium) and azurin (pink cartoon and gold copper - pdb code 3NP3)
Atypical interactions between GCPII and hydroxamate-based inhibitors
Authors: Novakova Zora , Barinkova Jitka , Slusher Barbara , Majer Pavel , Barinka Cyril
Published: November 2015

Human glutamate carboxypeptidase II (GCPII) is a zinc-dependent metallopeptidase implicated in diverse pathologies. GCPII-specific ligands can be used in diagnostic and therapeutic applications in prostate cancer and various neurological disorders [1]. Such ligands typically consist of a glutamate moiety linked to a zinc-binding group to ensure high specificity and affinity, respectively [2]. Hydroxamate functionality is one of the prominent zinc-binding functions used in the field, however, there are no structural data describing interactions between hydroxamates and GCPII. Here we report X-ray structures of six complexes between GCPII and hydroxamate-based inhibitors. Our structures reveal unexpected positioning of hydroxamates in the internal GCPII pocket that differs markedly from binding modes of matching prototypical GCPII inhibitors featuring different zinc-binding groups. They provide mechanistic explanation for prior structure-activity relationship studies and can be exploited for the structure-assisted design of novel GCPII-specific compounds.
Studies of Heat shock protein 27 Interactions
Authors: Aguda Adeleke , Nguyen Nham , Caner Sami , Moore Susan , Lelj-Garolla Barbara , Zoubeidi Amina , Gleave Martin , Brayer Gary
Published: November 2015

Human heat shock protein 27 (Hsp27) is an oligomeric and cell survival protein that has been associated with several cancers including prostrate and breast cancer. It's a known anti-apoptotic protein that functions as a molecular chaperone for several proteins. Hsp27 characteristically binds and stabilizes numerous partially unfolded proteins preventing their degradation, and has been shown to prevent actin polymerization in vitro. Several actin-binding residues involved in this interaction have been identified at the N-terminal loop and highly conserved alpha crystallin domains of Hsp27. Multiple assays have demonstrated that this hydrophobic actin-binding site is also involved in other protein binding. We therefore propose a common substrate-binding region on Hsp27 and present a model of Hsp27 binding to actin.
Molecular mechanisms of host cell manipulation by plant pathogens
Authors: Hughes Richard , King Stuart , Maqbool Abbas , McLellan Hazel , Bozkurt Tolga , Dagdas Yasin , Dong Suomeng , Birch Paul , Kamoun Sophien , Banfield Mark
Published: November 2015

An estimated 15% of global crop production is lost to pre-harvest disease every year. New ways to manage plant diseases are required. A mechanistic understanding of how plant pathogens re-program their hosts to enable colonisation may provide novel genetic or chemical opportunities to interfere with disease. One notorious plant parasite is the Irish potato famine pathogen Phytophthora infestans. This pathogen remains a considerable threat to potato/tomato crops today as the agent of late blight. Plant pathogens secrete effector proteins outside of and into plant cells to suppress host defences and manipulate cell physiology. Structural studies have provided insights into effector evolution and enabled experiments to probe function [1-3]. Crystal structures of 4 Phytophthora RXLR-type effectors, which are unrelated in primary sequence, revealed similarities in the fold of these proteins. This fold was proposed to act as a stable scaffold that supports diversification of effectors. Further, molecular modelling has enabled mapping of single-site variants responsible for specialisation of a Phytophthora Cystatin-like effector, revealing how effectors can adapt to new hosts after a "host jump". Structural studies describing how RXLR-effectors interact with host targets are lacking. We have used Y2H/co-IP studies to identify host proteins that interact with P. infestans effectors PexRD2 and PexRD54. PexRD2 interacts with MAPKKKe, a component of plant immune signalling pathways, and suppressed cell death activities of this protein. We used the structure of PexRD2 to design mutants that fail to interact with MAPKKKe, and no longer suppress cell-death activities. We found that PexRD54 interacts with potato homologues of the autophagy protein ATG8. We have obtained a crystal structure for PexRD54 in the presence of ATG8. We are now using X-ray scattering to verify the complex structure in solution prior to establishing the role of this interaction during infection.
Structural Biology of Mutant IDH2 Inhibition
Authors: DeLaBarre Byron , Wang Fang , Travins Jeremy , Gross Stefan , Artin Erin , Dang Lenny , Biller Scott , Yen Katherine
Published: November 2015

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases- 1 and -2 (IDH1, IDH2)[1]. These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite R (-)-2-hydroxyglutarate (2HG). 2HG is a potent inhibitor of DNA methylating enzymes such as TET2[2]. This suggests a connection between cancer related IDH mutations and aberrant epigenetics. As such, IDH represents an important new druggable target in the pursuit of novel cancer therapies. We have developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface[3]. While structures of IDH1 and IDH2 were known, this is the first ever structure of an inhibited IDH protein and shows a novel conformation of IDH2. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia (AML) cells in vitro. These data provide proof-of- concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.
Structural Studies of the Amyloid State of Proteins
Authors: Eisenberg David , Laganowsky Arthur , Liu Cong , Sawaya Michael , Whitelegge Julian , Zhao Minglei , Soriaga Angela , Landau Meytal , Teng Poh , Cascio Duilio , Jiang Lin , Sievers Stuart , Soragni Alice
Published: November 2015

Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. At the morphological level, these fibers appear similar and are termed "amyloid." We found that the adhesive segments of amyloid fibers are short protein sequences which form pairs of interdigitated, in-register beta sheets. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that in the neurodegenerative diseases, smaller, often transient and polymorphic oligomers are the toxic entities. We have identified a segment of the amyloid-forming protein, alphaB crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: beta-sheet-rich structure, cytotoxicity, and recognition by an anti-oligomer antibody. The X-ray-derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six anti-parallel, out-of-register protein strands, which we term a cylindrin. The cylindrin structure is compatible with sequence segments from the Abeta protein of Alzheimer's disease and from other amyloid proteins. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers, and are distinct in structure from amyloid fibrils.
SER-CAT Light When YOU Need It via automation & remote access
Authors: Rose John , Chrzas John , Fait Jim , Gonczy John , Fu "Zheng-Qing ""Albert""" , Jin Zhongmin , Salazar Rod , Chinte Unmesh , Kandavelu Palani , Wang Bi-Cheng
Published: November 2015

For the past 15 years, SER-CAT has been striving to provide its members with access to a "virtual beamline" which could be integrated into their daily workflow much like the X-ray lab down the hall. Working with Oceaneering Space Systems a conceptual design for automated data collection robot (ASTRO) was developed in 2000. In 2003, using funds from the Georgia Research Alliance, automation of the SER-CAT beamlines began with the installation of a highly modified Berkeley/ALS automounter (6 pucks) on beamline 22BM followed by a higher capacity system (15 pucks) installed on 22ID. SER-CAT automation also includes seamless integration with the SERGUI beamline control system, SER-CAT/UGA production of the tools (pucks, etc.) users need to ship crystals to the beamline and automated crystal screening and data reduction systems. Today over 95% of SER-CAT members routinely collect data remotely. Efficient and reliable automation has also allowed SER-CAT to offer two 12-hour data collection shifts per day with extended 16-hour/day on-site user support. An overview of SER-CAT beamline automation including robotics, beamline/experiment control, and automated data processing and structure determination will be described. Work supported by NIH NCRR (S10RR025528 & S10RR028976), SER-CAT Member Institutions, University of Georgia Research Foundation and the Georgia Research Alliance.
The Integration and Use of a Shutterless CMOS Detector at ALS Beamline 4.2.2
Authors: Nix Jay , Daly Robert
Published: November 2015

The Molecular Biology Consortium Beamline 4.2.2 at the Advanced Light Source has recently installed a new, high-speed, large surface-area CMOS detector for macromolecular crystallography by funds from NIH grant S10OD012073. The detector is 25 x 28 cm and has a readout speed greater than 25 frames per second. This allows shutterless operations and the collection of fine-sliced data. Presented is a description of the hardware and software integration of the detector into the controls system, and the implementation of a streamlined and intuitive collection interface. The interface is implemented in TCL/TK for integration into the beamline's custom Blu-Ice/EPICS environment and remote operations of the beamline with the new detector are routine. The increased sensitivity of the detector and shutterless operations allow for shorter exposure times per image, and up to 3X decrease in time per dataset. Optimum data collection and processing strategies to maximize the benefits of shutterless operations are discussed.
Mail-In Crystallography at the Canadian Light Source
Authors: Labiuk Shaunivan , Gorin James , Janzen Kathryn , Fodje Michel , Grochulski Pawel
Published: November 2015

The Canadian Macromolecular Crystallography Facility (CMCF) operates two crystallography beamlines, 08ID-1 and 08B1-1 (http://cmcf.lightsource.ca), at the Canadian Light Source (CLS). Normal access is through a traditional peer review process but commercial access is also welcome. Users apply for beamtime against their active proposals and may collect data in person or via remote access (Remote Control or Mail-In). Remote Control allows users to control the beamlines remotely from home institutions. Additionally, users with the highest peer review scores have the option of Mail-In beamtime, where data are collected by CMCF staff according to investigators' instructions. This method has proven very effective for several reasons. Data are collected by trained personnel experienced with the beamlines, resulting in high quality data. Travel expenses are eliminated. Data are collected around regular user shifts using the most appropriate beamline, reducing wasted beamtime and providing flexibility. The process is made extremely efficient when integrated with MxLIVE, the CMCF Laboratory Information Management System, since images can be viewed and output files downloaded almost immediately after data have been collected for the user. Four Mail-In shifts per week are typically supported during normal operations. In 2013 alone, over 430 datasets were collected for more than 20 investigators across Canada. At least 12 peer-reviewed articles appeared in 2013 containing Mail-In data, many in high-impact journals. Several of these results will be discussed.
Remote Access Capabilities at the GM/CA Beamlines at the APS
Authors: Ogata Craig , Stepanov Sergey , Pothineni Sudhir , Hilgart Mark , Sanishvili Ruslan , Nagarajan Venugopalan , Becker Michael , Makarov Oleg , Smith Janet , Fischetti Robert
Published: November 2015

The GM/CA facility consists of two undulator source beamlines and a bending magnet beamline at the Advanced Photon Source (APS). Access to the operation of these beamlines is accomplished through visits by investigators who are either on-site, remote or a combination of the two. In all modes of access, user operations are controlled by the experimenter. The control and capabilities of the GM/CA beamlines are identical for remote and on-site users. Remote access to the beamlines is through NX or Teamviewer to local computers [1]. Once communication has been established, experienced GM/CA experimenters are greeted by our familiar JBluIce, the graphical user interface/control program[2] responsible for all operations from sample handling through data collection and reduction. Although investigators always see a familiar interface, both software and hardware on the beamlines are continually improving. Recent hardware upgrades include a shift of the optical focusing mirrors on the ID-B beamline closer to the sample to provide a significant increase in flux, and installation of a new Pilatus3 6M detector on ID-D, the second undulator beamline. The JBluIce program has incorporated new detector controls for shutterless operation while continuing to expand the features of rastering, vector (helical) data collection, strategy tools and data analysis. These tools have been essential to investigators working on membrane crystal samples, e.g. GPCRs, as well as for samples that decay quickly or require data to be collected from multiple crystals. The presentation will provide an overview of beamline remote control as well as an update of the equipment that it operates at GM/CA.
Improved low-resolution crystallographic refinement with Phenix and Rosetta
Authors: Echols Nathaniel , DiMaio Frank , Headd Jeffrey , Terwilliger Thomas , Baker David , Adams Paul
Published: November 2015

Refinement of macromolecular structures against low-resolution crystallographic data is limited by the ability of current methods to arrive at a high-quality structure with realistic geometry. We have developed a new method for crystallographic refinement which combines the Rosetta sampling methodology and all atom energy function with likelihood-based reciprocal space refinement in Phenix, and find, on a test set of difficult low-resolution refinement cases, that models refined with the new method have significantly improved model geometry, and in most cases, lower free R factors and RMS deviation to the final model. Integration of the software packages additionally makes advanced sampling methods used in structure prediction and design available for crystallographic refinement and model-building, and also provides a strategy for improving the Rosetta force field for better agreement with experimental data.
CCP4 web services
Authors: Uski Ville , Ballard Charles , Keegan Ronan , Krissinel Eugene , Lebedev Andrey , Waterman David , Wojdyr Marcin
Published: November 2015

The CCP4 software suite [1] provides a comprehensive set of tools for use in the macromolecule structure solution process by X-ray crystallography. Traditionally, these tools have been run through the graphical interface or the command line on each user's personal workstation. Recently, some of the tools, including the molecular replacement pipelines Balbes [2] and MrBUMP [3] have been provided as web services in the Research Complex at Harwell. These pipelines can be especially useful in cases where there is low sequence identity between the target-structure sequence and that of its set of possible homologues. These services can be accessed through a web client, allowing one to submit molecular replacement jobs to our Linux cluster and view the results from these jobs. The molecular replacement pipelines are ideal candidates for web services, as they require installation and maintenance of large databases and benefit from parallel computing resources, provided by the cluster. Further plans for web services will be discussed. With ever-increasing mobility of scientific setups and the ubiquity of ultra-portable devices, there is a demand for a consistent framework of remote crystallographic computations and data maintenance. This framework is planned to include an interface for synchronising data with the facilities of Diamond Light Source, as well as with local CCP4 GUI-2 setups.
Low-Resolution Refinement of the DNA and Protein Complex Structures
Authors: Jiang Jiansheng , Jin Tengchuan , Xiao Tsan , Zhou Bing-Rui , Bai Yawen
Published: November 2015

Macromolecular complexes of protein and DNA are often resolved in a low resolution structure (3.0 angstroms or lower). Because nucleic acids suffer radiation damage more than amino acids, the resulting temperature factors for DNA are generally higher than those for protein. Recognition of DNA-specific interactions with protein is a challenge at low resolution. The use of low-resolution refinement ([1]) or the reference high resolution model could improve DNA densities. A number of DNA/protein and nucleosome complexes (i.e. RAGE-DNA [2], CENP-C-NCP[3]) that we have recently refined demonstrated the validation of these methods.
Photocrystallographic and kinetic studies of metastable linkage isomers
Authors: Hatcher Lauren , Raithby Paul
Published: November 2015

The ability of a molecular system to reversibly convert between two distinct states on photoactivation is desirable for many real-world applications, including photo-switchable device media. When induced in the solid-state, the process can be studied by crystallography and specifically photocrystallographic methods have contributed greatly to this diverse research area. The process of linkage isomerism has proved popular for photocrystallographic study. While the atomic rearrangements involved in the process are large enough to be determined from diffraction data, the changes are also moderate enough that crystal integrity is often maintained. Examples of species studied by these methods include nitrite, sulfur dioxide and nitrosyl coordination complexes [1]. Our research has focused on metal–nitrite complexes displaying nitro–nitrito isomerism and has aimed toward the rational design of new systems to undergo maximum conversion. Recently we have studied a selection of nickel–amine systems, including [Ni(Et4dien)(η2-O,ON)(η1-NO2)]. This complex shows diverse linkage isomeric behaviour on thermal and photochemical treatment [2], with 100% nitro–nitrito conversion achieved on excitation at λ = 500nm. The thermal and photochemical processes have been studied in detail [3] and the results of temperature studies, steady-state and pseudo-steady-state photocrystallographic experiments and solid-state kinetic studies are presented. The findings are discussed in relation to the key information they provide on the steric and kinetic factors that may influence the isomerisation process in the single-crystal.
Structural changes induced in crystals by the photocyclization and high pressure
Authors: Bakowicz Julia , Turowska-Tyrk Ilona
Published: November 2015

Photo-induced processes in crystals studied by means of X-ray structure analysis are the main subject of our interest. In particular, we monitor structural changes brought about by photochemical reactions. Nowadays, we have started to study influence of high pressure on them. In this poster, the results on monitoring structural changes in crystals of 2-tert-butylphenylphenylmethanone proceeding during the photocyclization reaction at ambient and high pressure will be presented. The studies demanded determination of structures of the pure reactant, pure product and many partly reacted crystals (Fig. 1) and were carried out at 0.1 MPa and 0.55, 1.27 and 1.50 GPa. Variations in the cell constants, geometry of the reaction centre and behavior of molecules during the phototransformation at various pressures will be presented. Several differences and similarities were observed. The studies provided knowledge on the path of the reaction in crystals and the influence of external factors, i.e. pressure, on it. Acknowledgments: The work was carried out within the grant 2011/01/D/ST5/02834 financed by the National Science Centre (Poland) and the fellowship co-financed by European Union within European Social Fund (Wroclaw University of Technology, Poland).
Structural dynamics of ionic materials mapped by femtosecond x-ray diffraction
Authors: Elsaesser Thomas
Published: November 2015

Relocation of electronic charge plays a key role for functional processes in condensed-phase molecular materials. X-ray diffraction with a femtosecond time resolution allows for spatially resolving transient atomic arrangements and charge distributions [1]. In particular, time-dependent spatial maps of electron density have been derived from x-ray powder diffraction patterns measured with a 100 fs time resolution. In this talk, new results on electron dynamics in transition metal complexes and on field-driven charge relocations in elementary ionic materials will be presented. Crystals containing a dense array of Fe(II)-tris(bipyridine) complexes and their PF6 counterions display pronounced changes of electron density that occur within the first 100 fs after two photon excitation of a small fraction of the complexes [2]. Electron density maps reveal a transfer of electronic charge from the Fe atoms and - so far unknown - from the PF6 counterions to the bipyridine units. The charge transfer displays pronounced Coulomb-mediated many-body features, affecting approximately 30 complexes around the directly excited one. As a second topic, electron relocations induced by strong external optical fields will be discussed [1,3]. This interaction mechanism allows for generating coherent superpositions of valence and conduction band quantum states and inducing fully reversible charge dynamics. While the materials LiBH4 and NaBH4 display electron relocations from the (BH4)- ions to the neighboring Li+ and Na+ ions, LiH exhibits an electron transfer from Li to H. The latter is a manifestation of electron correlations and in agreement with theoretical calculations.
Photochromism in a New Light: Temperature-Dependent in situ Photocrystallography
Authors: Cox Jordan , Walton Ian , Patel Dinesh , Xu Mengyang , Markelz Andrea , Benedict Jason
Published: November 2015

Organic photochromic molecules including diarylethenes are of particular interest for their potential applications in fields of high density optical data storage and light-activated switches, among many others. However, one of the limitations in diarylethene-based systems has been the low photoconversion observed in neat single crystals which is often less than 20%. The low conversion is typically believed to be the result of screening effects in which the photoisomerized molecules at the surface absorb incident light preventing full isomerization of the crystal. To assess the effect of screening on a model diarylethene system, photocrystallographic experiments on microcrystals of the compound were performed using synchrotron radiation at the Advanced Photon Source at Argonne National Labs. During the course of the study, we discovered that the photoconversion of the diarylethene crystals exhibits highly unusual temperature dependent behavior which is incongruent with current computational models of diarylethene photochemistry. Herein we report the first temperature-dependent `constant irradiation' in situ photocrystallography experiments performed on a photochromic system. Through the application of this technique, combined with spectroscopic analysis, we demonstrate that the steady-state population arising from the photo-cyclization reaction shows a temperature dependence which has been heretofore unobserved. Possible explanations for this anomalous behavior and its role in the photochemical reactivity of this and other diarylethene systems will be presented.
Phase Transition and Polymerization of Acetonitrile under High Pressure
Authors: Zheng Haiyan , Li Kuo , Cody George , Tulk Chris , Molaison Jamie , Yang Wenge , Ivanov Ilia , Guthrie Malcolm , Mao Ho-kwang
Published: November 2015

Successful application of high pressure on synthesis of organic polymer, including the conducting polymer and super hard materials depends on the knowledge of reaction mechanism. The evolution of crystal structure under high pressure especially the structure close to transition pressure is crucial to conclude the reaction mechanism. Nitriles represent a large class of interstellar molecules and are the potential source of amino acids. Understanding its behavior at extreme conditions has gained increasing attention recently. Acetonitrile (CH3CN), the simplest organic compound with C≡N triple bond, can act as a model system for studying the pressure induced polymerization. The phase transition of acetonitrile under high pressure has been studied extensively.[1-3] However, it is still controversial and there is no any detailed discussion about its polymerization mechanism under high pressure. Here, we report the in-situ high pressure Raman spectra and powder neutron diffraction results on CD3CN, which indicates a minor phase transition at 5 GPa. The neutron diffraction shows that CD3CN keeps the orthorhombic phase from 1.66 GPa to 20.58 GPa which is very close to the reaction pressure. The week hydrogen bonding CD...N arranges the molecule into 3-dimensional framework which can be treated as two sets of diamond like structures interpenetrating with each other. Interestingly, the observed N...D distance is 1.984 Å at 20.58 GPa, shorter than the van der Waals distance of N...H (2.75 Å) by 28%. The van der Waals separation is often taken as a reference distance for the molecular instability. Thus, a hydrogen transfer process during the polymerization can be concluded. This deduction is also supported by the solid state NMR and FTIR results of the recovered polymerized CH3CN (p-CH3CN) from high pressure. In addition, the atomic pair distribution function and Raman spectra indicate the p-CD3CN or p-CH3CN has a random packed layer structure with nano-graphene lattice.
In situ Raman spectroscopy of cubic boron nitride to 90 GPa and 800 K
Authors: Ono Shigeaki
Published: November 2015

Cubic boron nitride (c-BN) has some outstanding properties, such as hardness, chemical inertness, high temperature stability, and high thermal conductivity. The Raman spectrum of c-BN exhibits two intense lines at 1054 and 1305 cm-1 under ambient conditions, corresponding to the Brillouin zone center transverse optical (TO) and longitudinal optical (LO) modes, respectively. Previous studies have reported the pressure and temperature dependences of the frequency shift of the modes up to 40 GPa and 2300 K. The Raman line of the LO mode overlaps an intense Raman line of diamond at pressures higher than 3 GPa. Therefore, it is difficult to observe the LO line in high-pressure experiments using the diamond anvil cell. In contrast, previous studies proposed that the TO mode could be used as the pressure calibrant in diamond anvil cells under high pressure and temperature conditions. In this study, we used a diamond anvil cell high-pressure apparatus [1] combined with a Raman spectrometer system to investigate changes in the Raman line of c-BN. The use of a synchrotron radiation source made it possible to determine the precise pressure in the sample chamber. In this study, the temperature and pressure dependences of the Raman spectrum of the TO mode of cubic boron nitride were calibrated for applications to a Raman spectroscopy pressure sensor in optical cells to about 800 K and 90 GPa. A significant deviation from linearity of the pressure dependence is confirmed at pressures above 20 GPa. At ambient temperature, dv/dP slopes are 3.41 and 2.04 cm-1/GPa at 0 and 90 GPa, respectively. The pressure dependence does not significantly change with temperature, as determined from experiments conducted up to 800 K. At pressures above 90 GPa, the Raman spectrum of the TO mode cannot be observed because of an overlap of the signals of cubic boron nitride and diamond used as the anvils in the high-pressure cell.