On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination

Panjikar, Santosh; Parthasarathy, V.; Lamzin, Victor S.; Weiss, M.S.; Tucker, Paul A.

doi:10.1107/s0907444909029643

Cited by 163 publications

(157 citation statements)

References 49 publications

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“…The resulting electron density showed some interpretability for a small number of α-helices. Additional phase improvement was achieved using the MRSAD protocol of Auto-Rickshaw (49), and 40% of the model was built automatically. The model was then extended to ∼70% of the total number of residues by manual building into the resulting electron density map using COOT (50).…”

Section: Methodsmentioning

confidence: 99%

Stonefish toxin defines an ancient branch of the perforin-like superfamily

Ellisdon

Reboul

Panjikar

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack ComplexPerforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.H uman envenoming by the tropical stonefish (Synanceia horrida and related species) results in extreme pain, edema, hypotension, respiratory distress, and on rare occasions, death (1). The lethal factor in stonefish venom is an ∼150-kDa protein termed stonustoxin (SNTX), an unusual example of a vertebrate cytolytic protein complex (2). SNTX is a soluble heterodimeric assembly of two closely related proteins termed SNTX-α and -β that share sequence identity of ∼50% (3). With the exception of a C-terminal PRY SPla and the RYanodine Receptor (PRYSPRY) domain in each protein (4), SNTX shares no obvious sequence similarity to any structurally or functionally characterized molecule. SNTX induces species-specific hemolytic activity (2) by an apparent pore-forming mechanism (5). It induces platelet aggregation (6), and like the closely related Trachynilysin (from Synanceia trachynis), SNTX exhibits activity suggesting that it may function as a neurotoxin (7,8).Because eukaryote pore-forming toxins are relatively rare, we reasoned that SNTX might represent a new exemplar of a vertebrate pore-forming protein. Previous studies had shown that it was possible to purify and crystallize SNTX (9); however, no structure has been reported to date. Accordingly, to address the structural basis for SNTX activity, we determined its X-ray crystal structure.

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Section: Methodsmentioning

confidence: 99%

Stonefish toxin defines an ancient branch of the perforin-like superfamily

Ellisdon

Reboul

Panjikar

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…X-ray diffraction data were collected to a resolution of 2.5 Å at beamline MX2 at the Australian Synchrotron using the BlueIce data collection software 22 , integrated using XDS 23 and scaled and merged using Aimless 24 . Phasing was accomplished using SAD from a combination of Ta 6 Br 12 soaked and selenomethionine-substituted crystals [24][25][26] .…”

Section: Methods Summarymentioning

confidence: 99%

The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device

Busby

Panjikar

Landsberg

et al. 2013

Nature

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136

198

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The ABC toxin complexes (Tc) produced by certain bacteria are of interest due to their potent insecticidal activity 1,2 and potential role in human disease 3 . These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic 4 . The carboxy-terminal region of C is the main cytotoxic component 5 , and is poorly conserved between different Tcs. A general model of action has been proposed, in which the Tc binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells 5 . Tc complexes have been visualised using single particle electron microscopy 6,7 , but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, carboxy-terminal region of the C protein like the shell of an egg. The shell is decorated on one end with a -propeller domain, which mediates attachment of the B/C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example of a structure that contains RHS (rearrangement hot spot) repeats 8 , and illustrates a striking structural architecture that is likely conserved across both this widely distributed bacterial protein family and the related eukaryotic YD-repeatcontaining protein family, which includes the teneurins 9 . The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein encapsulation and delivery.ABC toxins were first identified, and have been best characterised, in the bacterium Photorhabdus luminescens 1 . However, the entomopathogenic bacterium Yersinia entomophaga contains a related Tc locus that includes an A component encoded by two ORFs (yenA1 and yenA2), a single B gene (yenB) and two C genes (yenC1 and yenC2), 10 the products of which associate independently with the A and B proteins, giving rise to two Tcs from one genetic locus. The C proteins of this and other Tcs are similar to the "polymorphic toxins" described by Zhang et al. 11 as they have a conserved RHS-repeat-containing amino-terminal region and a variable carboxyterminal region 10 . The carboxy-terminal regions (CTRs) of the Y. entomophaga C proteins are predicted to have different toxic activities: the C1 CTR is homologous to cytotoxic necrotising factor 1 (CNF1) from Escherichia coli 12 , whereas the C2 CTR is homologous to the deaminase YwqJ from Bacillus subtilis 13 . As in related complexes 3 , when the B subunit is co-expressed with either of the C subunits, C is cleaved at the boundary between th...

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“…The crystal structure was determined by the singlewavelength anomalous dispersion (SAD) method (52) using the Se-substituted crystals. The initial model was obtained using molecular replacement SAD (MRSAD) on the Auto-Rickshaw server (53). The structure of native ApcEΔ was determined by molecular replacement [Phaser in CCP4 (54) using ApcEΔ-Se as a starting model].…”

Section: Methodsmentioning

confidence: 99%

The terminal phycobilisome emitter, L _CM : A light-harvesting pigment with a phytochrome chromophore

Tang

Ding

Höppner

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Photosynthesis relies on energy transfer from light-harvesting complexes to reaction centers. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, attach to the membrane via the multidomain core-membrane linker, L CM . The chromophore domain of L CM forms a bottleneck for funneling the harvested energy either productively to reaction centers or, in case of light overload, to quenchers like orange carotenoid protein (OCP) that prevent photodamage. The crystal structure of the solubly modified chromophore domain from Nostoc sp. PCC7120 was resolved at 2.2 Å. Although its protein fold is similar to the protein folds of phycobiliproteins, the phycocyanobilin (PCB) chromophore adopts ZZZssa geometry, which is unknown among phycobiliproteins but characteristic for sensory photoreceptors (phytochromes and cyanobacteriochromes). However, chromophore photoisomerization is inhibited in L CM by tight packing. The ZZZssa geometry of the chromophore and π-π stacking with a neighboring Trp account for the functionally relevant extreme spectral red shift of L CM . Exciton coupling is excluded by the large distance between two PCBs in a homodimer and by preservation of the spectral features in monomers. The structure also indicates a distinct flexibility that could be involved in quenching. The conclusions from the crystal structure are supported by femtosecond transient absorption spectra in solution.cyanobacteria | phycobilisome | core-membrane linker | photosynthesis | crystal structure T he structural separation of light-harvesting and energy transduction processes allows photosynthetic organisms a flexible adaptation to the diverse light regimes present in terrestrial and aqueous habitats. In cyanobacteria and some phylogenetically related organisms that, together, provide a substantial proportion of global carbon fixation, light is harvested by hundreds of chromophores located in the peripheral antenna, namely, the phycobilisome (PBS). These excitations are then collected and transferred to the energy-transducing photosystems I (PSI) and PSII in the photosynthetic membrane via only two pigments, allophycocyanin B (AP-B) and the core-membrane linker (L CM ) (1-5). AP-B preferentially serves PSI (2); it is structurally similar to the bulk phycobiliproteins of the PBS (6). L CM (denoted as ApcE according to the gene by which it is encoded), preferentially serving PSII (5), is a more complex multidomain protein, with an N-terminal section that binds the phycocyanobilin (PCB) chromophore, and is homologous to phycobiliproteins but carries an additional loop that probably acts as a membrane anchor to PSII (4, 7-10). The Cterminal section consists of two to four repeats (depending on the PBS type) that are homologous to the N-terminal domain (Pfam PF00427) of structural PBS proteins, rod-linkers [Protein Data Bank (PDB) ID codes 3OSJ and 3OHW], and considered to organize the complex PBS core (9). They are probably also the loci for phosphorylation (11).Both AP-B and L CM contain the same PCB chromophores...

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On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination

Cited by 163 publications

References 49 publications

Stonefish toxin defines an ancient branch of the perforin-like superfamily

Stonefish toxin defines an ancient branch of the perforin-like superfamily

The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device

The terminal phycobilisome emitter, L _CM : A light-harvesting pigment with a phytochrome chromophore

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On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination

Cited by 163 publications

References 49 publications

Stonefish toxin defines an ancient branch of the perforin-like superfamily

Stonefish toxin defines an ancient branch of the perforin-like superfamily

The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device

The terminal phycobilisome emitter, L CM : A light-harvesting pigment with a phytochrome chromophore

Contact Info

Product

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The terminal phycobilisome emitter, L _CM : A light-harvesting pigment with a phytochrome chromophore