Automatic protein structure solution from weak X-ray data

Skubák, Pavol; Pannu, Navraj S.

doi:10.1038/ncomms3777

Cited by 270 publications

(193 citation statements)

References 23 publications

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“…These sites were refined and experimental phases to 3.4 Å were calculated using the multiple anomalous dispersion (MAD) procedure in autoSHARP. These phases were further improved using Crank2 (Skubak and Pannu 2013), incorporating model building and refinement with Buccaneer (Cowtan 2006) and REFMAC5 (Murshudov et al 2011). The initial model produced was positioned in the native data set with Phaser (McCoy et al 2007).…”

Section: Maelstrom Purificationmentioning

confidence: 99%

Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists

Chen

Campbell

Pandey

et al. 2015

RNA

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Piwi-interacting RNAs (piRNAs) guide Piwi argonautes to their transposon targets for silencing. The highly conserved protein Maelstrom is linked to both piRNA biogenesis and effector roles in this pathway. One defining feature of Maelstrom is the predicted MAEL domain of unknown molecular function. Here, we present the first crystal structure of the MAEL domain from Bombyx Maelstrom, which reveals a nuclease fold. The overall architecture resembles that found in Mg 2+ -or Mn 2+ -dependent DEDD nucleases, but a clear distinguishing feature is the presence of a structural Zn 2+ ion coordinated by the conserved ECHC residues. Strikingly, metazoan Maelstrom orthologs across the animal kingdom lack the catalytic DEDD residues, and as we show for Bombyx Maelstrom are inactive as nucleases. However, a MAEL domain-containing protein from amoeba having both sequence motifs (DEDD and ECHC) is robustly active as an exoribonuclease. Finally, we show that the MAEL domain of Bombyx Maelstrom displays a strong affinity for single-stranded RNAs. Our studies suggest that the ancient MAEL nuclease domain evolved to function as an RNA-binding module in metazoan Maelstrom.

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Section: Maelstrom Purificationmentioning

confidence: 99%

Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists

Chen

Campbell

Pandey

et al. 2015

RNA

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“…The structure of LOR_107d was solved with the automated structure solving pipeline CRANK2 (28) using data from the SeMet crystal. A total of 20 selenium sites were identified and the initial model contained 839 residues out of 1000.…”

Section: Structure Determination and Refinementmentioning

confidence: 99%

Structure–function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism

Ulaganathan

Helbert

Kopel

et al. 2018

Journal of Biological Chemistry

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Ulvan is a major cell wall component of green algae of the genus Ulva and some marine bacteria encode enzymes that can degrade this polysaccharide. The first ulvan degrading lyases have been recently characterized and several putative ulvan lyases have been recombinantly expressed, confirmed as ulvan lyases and partially characterized. Two families of ulvan degrading lyases, PL24 and PL25, have recently been established. The PL24 lyase LOR_107 from the bacterial Alteromonadales sp. strain LOR degrades ulvan endolytically, cleaving the bond at the C4 of a glucuronic acid. However, the mechanism and LOR_107 structural features involved are unknown. We present here the crystal structure of LOR_107, representing the first PL24 family structure. We found that LOR_107 adopts a seven-bladed β-propeller fold with a deep canyon on one side of the protein. Comparative sequence analysis revealed a cluster of conserved residues within this canyon, and site-directed mutagenesis disclosed several residues essential for catalysis. We also found that LOR_107 uses the His/Tyr catalytic mechanism, common to several PL families. We captured a tetrasaccharide substrate in the structures of two inactive mutants, which indicated a two-step binding event, with the first substrate interaction near the top of the canyon coordinated by Arg-320, followed by sliding of the substrate into the canyon toward the active-site residues. Surprisingly, the LOR_107 structure was very similar to that of PL25 family PLSV_3936, despite only ~14% sequence identity between the two enzymes. On the basis of our structural and mutational analyses, we propose a catalytic mechanism for LOR_107 that differs from the typical His/Tyr mechanism.Ulvan is one of the two major cell wall components of marine green algae (genus Ulva and Enteromorpha). It is a complex sulfated polysaccharide composed mainly of 3-sulfated rhamnose (Rha3S), glucuronic acid (GlcA), iduronic acid (IdoA) and xylose (1). The common disaccharide repetitive units within the ulvan polysaccharides are [→4)-β-D-GlcA-(1→4)-α-LRha3S-(1→] called type A ulvanobiourinic-3-sulfate (A 3S ) and [→4)-α-L-IdoA-(1→4)-α-LRha3S(1→] called type B ulvanobiouronic-3-sulfate (B 3S ) (1) (Schema 1). The presence of iduronic acid and sulfated rhamnose differentiates ulvan from other polysaccharides of marine origin and displays similarity with mammalian glycosaminoglycans such as chondroitin sulfate and hyaluronic acid. This distinctive chemical feature makes ulvan an attractive candidate for various biomedical, nanobiotechnological and drug delivery applications (2-6).Polysaccharides containing uronic acid sugars can be degraded by enzymes utilizing a β-http://www.jbc.org/cgi/doi/10.1074/jbc.RA117. (PLs). They utilize a β-elimination mechanism to cleave the oxygenaglycone bond by abstracting the C5 proton, which results in the formation of an unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (ΔUA) at the non-reducing end of the oligosaccharide product (7). These enzymes are presently classified into 26 s...

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“…The SAD phasing method can be challenging if the anomalous signal-to-noise ratio is low 10,11 . The magnitudes of the anomalous differences between Bijvoet pairs of reflections depend on the types and numbers of anomalously scattering atoms in the structure and the wavelength of data collection, and their accuracy depends on the details of data collection, particularly the number of times each X-ray reflection is measured.…”

mentioning

confidence: 99%

Macromolecular X-ray structure determination using weak, single-wavelength anomalous data

et al. 2014

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A likelihood-based method for determining the sub-structure of anomalously-scattering atoms in macromolecular crystals can allow successful structure determination by single-wavelength anomalous diffraction (SAD) X-ray analysis with weak anomalous signal. Along with use of partial models and electron density maps in searches for anomalously-scattering atoms, testing of alternative values of parameters, and parallelized automated model-building, this method has the potential for extending the applicability of the SAD method in challenging cases.

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Automatic protein structure solution from weak X-ray data

Cited by 270 publications

References 23 publications

Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists

Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists

Structure–function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism

Macromolecular X-ray structure determination using weak, single-wavelength anomalous data

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