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Eleonore von Castelmur,
Johan Strümpfer,
Barbara Franke,
Julijus Bogomolovas,
Sonia Barbieri,
Hiroshi Qadota,
Petr V Konarev,
Dmitri I Svergun,
Siegfried Labeit,
Guy M Benian,
Klaus Schulten, Olga Mayans
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ABSTRACT: Titin-like kinases are an important class of cytoskeletal kinases that intervene in the response of muscle to mechanical stimulation, being central to myofibril homeostasis and development. These kinases exist in autoinhibited states and, allegedly, become activated during muscle activity by the elastic unfolding of a C-terminal regulatory segment (CRD). However, this mechano-activation model remains controversial. Here we explore the structural, catalytic, and tensile properties of the multidomain kinase region of Caenorhabditis elegans twitchin (Fn(31)-Nlinker-kinase-CRD-Ig(26)) using X-ray crystallography, small angle X-ray scattering, molecular dynamics simulations, and catalytic assays. This work uncovers the existence of an inhibitory segment that flanks the kinase N-terminally (N-linker) and that acts synergistically with the canonical CRD tail to silence catalysis. The N-linker region has high mechanical lability and acts as the primary stretch-sensor in twitchin kinase, while the CRD is poorly responsive to pulling forces. This poor response suggests that the CRD is not a generic mechanosensor in this kinase family. Instead, the CRD is shown here to be permissive to catalysis and might protect the kinase active site against mechanical damage. Thus, we put forward a regulatory model where kinase inhibition results from the combined action of both N- and C-terminal tails, but only the N-terminal extension undergoes mechanical removal, thereby affording partial activation. Further, we compare invertebrate and vertebrate titin-like kinases and identify variations in the regulatory segments that suggest a mechanical speciation of these kinase classes.
Proceedings of the National Academy of Sciences 08/2012; 109(34):13608-13. · 9.68 Impact Factor
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ABSTRACT: Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the characterization of Ig domains of intracellular origin--in particular, modular components of the titin myofilament. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization.
Protein Engineering Design and Selection 02/2012; 25(5):205-12. · 2.94 Impact Factor
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ABSTRACT: MuRFs, brief for muscle specific RING finger proteins, correspond to a subfamily of the TRIM/RBCC protein family. Here, we review recent progress on the structural biology of MuRF1, the MuRF family member being most clearly associated with muscle diseases. The emerging understanding of the structural biology of MuRFs and their interaction with their numerous myocellular proteins, at least in part representing ubiquitination targets for MuRFs, is likely to provide future rationales to modulate their activity, thus affecting their roles in muscle disease progression.
Advances in experimental medicine and biology 01/2012; 770:119-29. · 1.09 Impact Factor
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ABSTRACT: Titin is a giant filamentous protein of striated muscle composed of >300 immunoglobulin-like domains linked in tandem. Here, we demonstrate that a six-immunoglobulin fragment of titin carrying a poly-histidine tag forms a protein "brush" on liposomes containing metallochelating lipids. These specimens might allow the direct visualization by cryo-EM of frozen hydrated and unstained titin chains with preserved architectural features.
Journal of Bioscience and Bioengineering 05/2011; 112(2):178-9. · 1.79 Impact Factor
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ABSTRACT: The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.
Nano Letters 10/2010; 10(11):4533-7. · 13.20 Impact Factor
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ABSTRACT: Titin is a large intrasarcomeric protein that, among its many roles in muscle, is thought to modulate the in vivo assembly of the myosin motor filament. This is achieved through the molecular template properties of its A-band region, which is composed of fibronectin type III (FnIII) and immunoglobulin (Ig) domains organized into characteristic 7-domain (D-zone) and 11-domain (C-zone) superrepeats. Currently, there is little knowledge on the structural details of this region of titin. Here we report the conformational characterization of three FnIII tandems, A77-A78, A80-A82, and A84-A86, which are components of the representative fourth C-zone superrepeat. The structure of A77-A78 has been elucidated by X-ray crystallography to 1.65 A resolution, while low-resolution models of A80-A82 and A84-A86 have been calculated using small-angle X-ray scattering. A77-A78 adopts an extended "up-down" domain arrangement, where domains are connected by a hydrophilic three-residue linker sequence. The linker is embedded in a rich network of polar contacts at the domain interface that results in a stiff molecular conformation. The models of A80-A82 and A84-A86, which contain hydrophobic six-residue-long interdomain linkers, equally showed elongated molecular shapes, but with slightly coiled or zigzagged conformations. Small-angle X-ray scattering data further suggested that the long linkers do not result in a noticeable increase in molecular flexibility but lead to semibent domain arrangements. Our findings indicate that the structural characteristics of FnIII tandems from A-band titin contrast markedly with those of poly-Ig tandems from the elastic I-band, which exhibit domain interfaces depleted of interactions and compliant conformations. Furthermore, the analysis of sequence conservation in FnIII domains from A-band titin points to the existence of conformationally defined interfaces at specific superrepeat positions, possibly leading to a periodic and locally ordered architecture supporting the molecular scaffold properties of this region of titin.
Journal of Molecular Biology 09/2010; 401(5):843-53. · 4.00 Impact Factor
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ABSTRACT: The protein titin functions as a mechanical spring conferring passive elasticity to muscle. Force spectroscopy studies have shown that titin exhibits several regimes of elasticity. Disordered segments bring about a soft, entropic spring-type elasticity; secondary structures of titin's immunoglobulin-like (Ig-) and fibronectin type III-like (FN-III) domains provide a stiff elasticity. In this study, we demonstrate a third type of elasticity due to tertiary structure and involving domain-domain interaction and reorganization along the titin chain. Through 870 ns of molecular dynamics simulations involving 29,000-635,000 atom systems, the mechanical properties of a six-Ig domain segment of titin (I65-I70), for which a crystallographic structure is available, are probed. The results reveal a soft tertiary structure elasticity. A remarkably accurate statistical mechanical description for this elasticity is derived and applied. Simulations also studied the stiff, secondary structure elasticity of the I65-I70 chain due to the unraveling of its domains and revealed how force propagates along the chain during the secondary structure elasticity response.
Biophysical Journal 03/2010; 98(6):1085-95. · 3.65 Impact Factor
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ABSTRACT: Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (ssAnPRT) is encoded by the sstrpD gene and catalyzes the reaction of anthranilate (AA) with a complex of Mg(2+) and 5'-phosphoribosyl-alpha1-pyrophosphate (Mg.PRPP) to N-(5'-phosphoribosyl)-anthranilate (PRA) and pyrophosphate (PP(i)) within tryptophan biosynthesis. The ssAnPRT enzyme is highly thermostable (half-life at 85 degrees C = 35 min) but only marginally active at ambient temperatures (turnover number at 37 degrees C = 0.33 s(-1)). To understand the reason for the poor catalytic proficiency of ssAnPRT, we have isolated from an sstrpD library the activated ssAnPRT-D83G + F149S double mutant by metabolic complementation of an auxotrophic Escherichia coli strain. Whereas the activity of purified wild-type ssAnPRT is strongly reduced in the presence of high concentrations of Mg(2+) ions, this inhibition is no longer observed in the double mutant and the ssAnPRT-D83G single mutant. The comparison of the crystal structures of activated and wild-type ssAnPRT shows that the D83G mutation alters the binding mode of the substrate Mg.PRPP. Analysis of PRPP and Mg(2+)-dependent enzymatic activity indicates that this leads to a decreased affinity for a second Mg(2+) ion and thus reduces the concentration of enzymes with the inhibitory Mg(2).PRPP complex bound to the active site. Moreover, the turnover number of the double mutant ssAnPRT-D83G + F149S is elevated 40-fold compared to the wild-type enzyme, which can be attributed to an accelerated release of the product PRA. This effect appears to be mainly caused by an increased conformational flexibility induced by the F149S mutation, a hypothesis which is supported by the reduced thermal stability of the ssAnPRT-F149S single mutant.
Biochemistry 05/2009; 48(23):5199-209. · 3.42 Impact Factor
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ABSTRACT: The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.
Biochemistry 10/2008; 47(40):10722-30. · 3.42 Impact Factor
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ABSTRACT: Low-resolution diffraction data (resolution below 12 angstroms) from crystals of a filamentous six-Ig fragment of titin, I65-I70, were used in ab initio phasing with the aim of calculating its lattice packing and molecular envelope. Filamentous molecules, characterized by marked anisometry and idiosyncratic crystal lattices, have not been addressed before using this methodology. In this study, low-resolution phasing (19-122 angstroms) successfully identified the region of the unit cell occupied by the molecule. Phase extension to a higher resolution (12 angstroms) yielded regions of high density that corresponded either to the positions of individual Ig domains or to zones of dense intermolecular contacts, hindering the identification of individual domains and the interpretation of electron-density maps in terms of a molecular model. This problem resulted from the acutely uneven packing of the molecules in the crystal and it was further accentuated by the presence of partially disordered regions in the molecule. Addition of low-resolution reflections with phases computed ab initio to those obtained experimentally using MIRAS improved the initial electron-density maps of the atomic model, demonstrating the generic utility of low-resolution phases for the structure-elucidation process, even when individual molecules cannot be resolved in the lattice.
Acta Crystallographica Section D Biological Crystallography 06/2008; 64(Pt 5):478-86. · 12.62 Impact Factor
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ABSTRACT: The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 microM and 20 microM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.
Journal of Molecular Biology 03/2008; 376(2):506-16. · 4.00 Impact Factor
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ABSTRACT: Myofibril elasticity, critical to muscle function, is dictated by the intrasarcomeric filament titin, which acts as a molecular spring. To date, the molecular events underlying the mechanics of the folded titin chain remain largely unknown. We have elucidated the crystal structure of the 6-Ig fragment I65-I70 from the elastic I-band fraction of titin and validated its conformation in solution using small angle x-ray scattering. The long-range properties of the chain have been visualized by electron microscopy on a 19-Ig fragment and modeled for the full skeletal tandem. Results show that conserved Ig-Ig transition motifs generate high-order in the structure of the filament, where conformationally stiff segments interspersed with pliant hinges form a regular pattern of dynamic super-motifs leading to segmental flexibility in the chain. Pliant hinges support molecular shape rearrangements that dominate chain behavior at moderate stretch, whereas stiffer segments predictably oppose high stretch forces upon full chain extension. There, librational entropy can be expected to act as an energy barrier to prevent Ig unfolding while, instead, triggering the unraveling of flanking springs formed by proline, glutamate, valine, and lysine (PEVK) sequences. We propose a mechanistic model based on freely jointed rigid segments that rationalizes the response to stretch of titin Ig-tandems according to molecular features.
Proceedings of the National Academy of Sciences 02/2008; 105(4):1186-91. · 9.68 Impact Factor
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ABSTRACT: The giant protein titin, which is responsible for passive elasticity in muscle fibers, is built from approximately 300 regular immunoglobulin-like (Ig) domains and FN-III repeats. While the soft elasticity derived from its entropic regions, as well as the stiff mechanical resistance derived from the unfolding of the secondary structure elements of Ig- and FN-III domains have been studied extensively, less is known about the mechanical elasticity stemming from the orientation of neighboring domains relative to each other. Here we address the dynamics and energetics of interdomain arrangement of two adjacent Ig-domains of titin, Z1, and Z2, using molecular dynamics (MD) simulations. The simulations reveal conformational flexibility, due to the domain-domain geometry, that lends an intermediate force elasticity to titin. We employ adaptive biasing force MD simulations to calculate the energy required to bend the Z1Z2 tandem open to identify energetically feasible interdomain arrangements of the Z1 and Z2 domains. The finding is cast into a stochastic model for Z1Z2 interdomain elasticity that is generalized to a multiple domain chain replicating many Z1Z2-like units and representing a long titin segment. The elastic properties of this chain suggest that titin derives so-called tertiary structure elasticity from bending and twisting of its domains. Finally, we employ steered molecular dynamics simulations to stretch individual Z1 and Z2 domains and characterize the so-called secondary structure elasticity of the two domains. Our study suggests that titin's overall elastic response at weak force stems from a soft entropic spring behavior (not described here), from tertiary structure elasticity with an elastic spring constant of approximately 0.001-1 pN/A and, at strong forces, from secondary structure elasticity.
Biophysical Journal 10/2007; 93(5):1719-35. · 3.65 Impact Factor
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ABSTRACT: Titin forms an intrasarcomeric filament system in vertebrate striated muscle, which has elastic and signaling properties and is thereby central to mechanotransduction. Near its C-terminus and directly preceding a kinase domain, titin contains a conserved pattern of Ig and FnIII modules (Ig(A168)-Ig(A169)-FnIII(A170), hereby A168-A170) that recruits the E3 ubiquitin-ligase MuRF-1 to the filament. This interaction is thought to regulate myofibril turnover and the trophic state of muscle. We have elucidated the crystal structure of A168-A170, characterized MuRF-1 variants by circular dichroism (CD) and SEC-MALS, and studied the interaction of both components by isothermal calorimetry, SPOTS blots, and pull-down assays. This has led to the identification of the molecular determinants of the binding. A168-A170 shows an extended, rigid architecture, which is characterized by a shallow surface groove that spans its full length and a distinct loop protrusion in its middle point. In MuRF-1, a C-terminal helical domain is sufficient to bind A168-A170 with high affinity. This helical region predictably docks into the surface groove of A168-A170. Furthermore, pull-down assays demonstrate that the loop protrusion in A168-A170 is a key mediator of MuRF-1 recognition. Our findings indicate that this region of titin could serve as a target to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover, where binding of small molecules to its distinctive structural features could block MuRF-1 access.
The FASEB Journal 06/2007; 21(7):1383-92. · 5.71 Impact Factor
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ABSTRACT: The nucleoplasmic protein, Lamina-associated polypeptide (LAP) 2α, is one of six alternatively spliced products of the LAP2gene, which share a common N-terminal region. In contrast to the other isoforms, which also share most of their C termini,
LAP2α has a large unique C-terminal region that contains binding sites for chromatin, A-type lamins, and retinoblastoma protein.
By immunoprecipitation analyses of LAP2α complexes from cells expressing differently tagged LAP2α proteins and fragments,
we demonstrate that LAP2α forms higher order structures containing multiple LAP2α molecules in vivo and that complex formation is mediated by the C terminus. Solid phase binding assays using recombinant and in vitro translated LAP2α fragments showed direct interactions of LAP2α C termini. Cross-linking of LAP2α complexes and multiangle
light scattering of purified LAP2α revealed the existence of stable homo-trimers in vivo and in vitro. Finally, we show that, in contrast to the LAP2α-lamin A interaction, its self-association is not affected by a disease-linked
single point mutation in the LAP2α C terminus.
Journal of Biological Chemistry 03/2007; 282(9):6308-6315. · 4.77 Impact Factor
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ABSTRACT: Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.
Structure 10/2006; 14(9):1437-47. · 6.35 Impact Factor
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ABSTRACT: The metabolic synthesis and degradation of essential nucleotide compounds are primarily carried out by phosphoribosyltransferases (PRT) and nucleoside phosphorylases (NP), respectively. Despite the resemblance of their reactions, five classes of PRTs and NPs exist, where anthranilate PRT (AnPRT) constitutes the only evolutionary link between synthesis and degradation processes. We have characterized the active site of dimeric AnPRT from Sulfolobus solfataricus by elucidating crystal structures of the wild-type enzyme complexed to its two natural substrates anthranilate and 5-phosphoribosyl-1-pyrophosphate/Mg(2+). These bind into two different domains within each protomer and are brought together during catalysis by rotational domain motions as shown by small angle x-ray scattering data. Steady-state kinetics of mutated AnPRT variants address the role of active site residues in binding and catalysis. Results allow the comparative analysis of PRT and pyrimidine NP families and expose related structural motifs involved in nucleotide/nucleoside recognition by these enzyme families.
Journal of Biological Chemistry 08/2006; 281(30):21410-21. · 4.77 Impact Factor
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ABSTRACT: Self-assembly via coiled-coil domains (CC) is crucial for the regulation of the dystrophia myotonica kinase (DMPK) -related family of kinases. These CC domains are thought to form dimeric arrangements and thus to mediate dimerization in these enzymes. Using size exclusion chromatography combined with multiangle static light scattering, we analyzed the oligomeric state of DMPK as well as that of a truncated variant lacking the CC fraction. Remarkably, both forms were found to assemble into robust dimers. In contrast, the CC domain in isolation yielded trimeric assemblies, indicating that the oligomerization properties of CC domains from this kinase family are more diversified than anticipated. The crystal structure of this CC has been elucidated to 1.6 angstroms resolution and its properties in solution established using sedimentation equilibrium and thermal denaturation. These data show that, contrary to expectations, the self-assembly of DMPK is not dictated by the association properties of its CC domain. Instead, it appears to be driven by sequence segments flanking both N and C termini of the catalytic kinase fraction, as suggested by models of head-to-head dimers based on small angle X-ray scattering data. Our findings support a shared pattern of assembly across DMPK, ROCKs, and MRCK members of this family.
The FASEB Journal 07/2006; 20(8):1142-51. · 5.71 Impact Factor
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ABSTRACT: The cellular function of the giant protein titin in striated muscle is a major focus of scientific attention. Particularly, its role in passive mechanics has been extensively investigated. In strong contrast, the structural details of this filament are very poorly understood. To date, only a handful of atomic models from single domain components have become available and data on poly-constructs are limited to scarce SAXS analyses. In this study, we examine the molecular parameters of poly-Ig tandems from I-band titin relevant to muscle elasticity. We revisit conservation patterns in domain and linker sequences of I-band modules and interpret these in the light of available atomic structures of Ig domains from muscle proteins. The emphasis is placed on features expected to affect inter-domain arrangements. We examine the overall conformation of a 6Ig fragment, I65-I70, from the skeletal I-band of soleus titin using SAXS and electron microscopy approaches. The possible effect of highly conserved glutamate groups at the linkers as well as the ionic strength of the medium on the overall molecular parameters of this sample is investigated. Our findings indicate that poly-Ig tandems from I-band titin tend to adopt extended arrangements with low or moderate intrinsic flexibility, independently of the specific features of linkers or component Ig domains across constitutively- and differentially-expressed tandems. Linkers do not appear to operate as free hinges so that lateral association of Ig domains must occur infrequently in samples in solution, even that inter-domain sequences of 4-5 residues length would well accommodate such geometry. It can be expected that this principle is generally applicable to all Ig-tandems from I-band titin.
Journal of Muscle Research and Cell Motility 02/2005; 26(6-8):355-65. · 1.98 Impact Factor
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ABSTRACT: The coiled-coil domain of dystrophia myotonica protein kinase (DMPK) has been cloned, overexpressed, purified and crystallized. Two crystal forms have been obtained that belong to space groups P3 and P2(1)2(1)2(1) and diffract to 2.4 and 1.6 A resolution, respectively. Experimental phases were obtained by MAD from an SeMet derivative. The location of selenium sites used molecular-replacement phases obtained from search models lacking sequence similarity with the coiled-coil under study. Both crystal forms contain three polypeptide chains in the asymmetric unit.
Acta Crystallographica Section D Biological Crystallography 01/2005; 60(Pt 12 Pt 2):2336-9. · 12.62 Impact Factor
