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Instruments / Technical Area	Publication Details	Published
	IceBear : an intuitive and versatile web application for research-data tracking from crystallization experiment to PDB deposition Ed Daniel , Mirko M. Maksimainen , Neil Smith , Ville Ratas , Ekaterina Biterova , Sudarshan N. Murthy , M. Tanvir Rahman , Tiila-Riikka Kiema , Shruthi Sridhar , Gabriele Cordara , Subhadra Dalwani , Rajaram Venkatesan , Jaime Prilusky , Orly Dym , Lari Lehtio , M. Kristian Koski , Alun W. Ashton , Joel L. Sussman , Rikkert K. Wierenga Acta Crystallographica Section D Structural Biology 77 DOI: 10.1107/S2059798320015223 Open Access Show Abstract ▼ Abstract: The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank.	Feb 2021
I03-Macromolecular Crystallography	Crystallographic binding studies of rat peroxisomal multifunctional enzyme type 1 with 3-ketodecanoyl-CoA: capturing active and inactive states of its hydratase and dehydrogenase catalytic sites Shruthi Sridhar , Werner Schmitz , J. Kalervo Hiltunen , Rajaram Venkatesan , Ulrich Bergmann , Tiila-Riikka Kiema , Rikkert K. Wierenga Acta Crystallographica Section D Structural Biology 76, 1256 - 1269 DOI: 10.1107/S2059798320013819 Diamond Proposal Number(s): [26302, 24732] Show Abstract ▼ Abstract: The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the β-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD+-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 Å resolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD+ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.	Dec 2020
I04-Macromolecular Crystallography	Insights into the stability and substrate specificity of the E. coli aerobic β-oxidation trifunctional enzyme complex Shiv K. Sah-teli , Mikko J. Hynonen , Ramita Sulu , Subhadra Dalwani , Werner Schmitz , Rik. K. Wierenga , Rajaram Venkatesan Journal Of Structural Biology DOI: 10.1016/j.jsb.2020.107494 Diamond Proposal Number(s): [14794] Show Abstract ▼ Abstract: Degradation of fatty acids by the β-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In E. coli, the last three steps of the β-oxidation are catalyzed by two heterotetrameric α2β2 enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α-subunit of TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the β-subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of Pseudomans fragi and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-β is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain 2E-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of β-oxidation in E. coli.	Mar 2020
Krios I-Titan Krios I at Diamond	A novel druggable interprotomer pocket in the capsid of rhino- and enteroviruses Rana Abdelnabi , James A. Geraets , Yipeng Ma , Carmen Mirabelli , Justin W. Flatt , Ausra Domanska , Leen Delang , Dirk Jochmans , Timiri Ajay Kumar , Venkatesan Jayaprakash , Barij Nayan Sinha , Pieter Leyssen , Sarah Butcher , Johan Neyts Plos Biology DOI: 10.1371/journal.pbio.3000281 Diamond Proposal Number(s): [1973] Open Access Show Abstract ▼ Abstract: Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are “capsid binders” that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo–electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.	Jun 2019
B21-High Throughput SAXS	Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic beta-oxidation trifunctional enzyme complexes Shiv Sah-teli , Mikko J. Hynönen , Werner Schmitz , James A. Geraets , Jani Seitsonen , Jan Skov Pedersen , Sarah J. Butcher , Rik .k. Wierenga , Rajaram Venkatesan Biochemical Journal DOI: 10.1042/BCJ20190314 Diamond Proposal Number(s): [14794] Open Access Show Abstract ▼ Abstract: The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid β-oxidation cycle. Two TFEs are present in Escherichia coli , EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane bound complex. Systematic enzyme kinetic studies show that anEcTFE has preference for medium and long chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.	Jun 2019
I04-Macromolecular Crystallography	Insights into mitochondrial fatty acid synthesis from the structure of heterotetrameric 3-ketoacyl-ACP reductase/3R-hydroxyacyl-CoA dehydrogenase Rajaram Venkatesan , Shiv K. Sah-teli , Luqman O. Awoniyi , Guangyu Jiang , Piotr Prus , Alexander J. Kastaniotis , J. Kalervo Hiltunen , Rik K. Wierenga , Zhijun Chen Nature Communications 5, 4805 DOI: 10.1038/ncomms5805 PMID: 25203508 Diamond Proposal Number(s): [8030] Show Abstract ▼ Abstract: Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory growth in yeast and mammalian embryonic survival. The human 3-ketoacyl-acyl carrier protein (ACP) reductase (KAR) of mtFAS is a heterotetrameric á2â2-assembly composed of 17â-hydroxysteroid dehydrogenase type-8 (HSD17B8, á-subunit) and carbonyl reductase type-4 (CBR4, â-subunit). Here we provide a structural explanation for the stability of the heterotetramer from the crystal structure with NAD(+) and NADP(+) bound to the HSD17B8 and CBR4 subunits, respectively, and show that the catalytic activity of the NADPH- and ACP-dependent CBR4 subunit is crucial for a functional HsKAR. Therefore, mtFAS is NADPH- and ACP dependent, employing the 3R-hydroxyacyl-ACP intermediate. HSD17B8 assists in the formation of the competent HsKAR assembly. The intrinsic NAD(+)- and CoA-dependent activity of the HSD17B8 subunit on the 3R-hydroxyacyl-CoA intermediates may indicate a role for this subunit in routing 3R-hydroxyacyl-CoA esters, potentially arising from the metabolism of unsaturated fatty acids, into the mitochondrial â-oxidation pathway.	Sep 2014

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