MBP-binding DARPins facilitate the crystallization of an MBP fusion protein

The production of high-quality crystals is the main bottleneck in determining the structures of proteins using X-ray crystallography. In addition to being recognized as a very effective solubility-enhancing fusion partner,Escherichia colimaltose-binding protein (MBP) has also been successfully employed as a `fixed-arm' crystallization chaperone in more than 100 cases. Here, it is reported that designed ankyrin-repeat proteins (DARPins) that bind with high affinity to MBP can promote the crystallization of an MBP fusion protein when the fusion protein alone fails to produce diffraction-quality crystals. As a proof of principle, three different co-crystal structures of MBP fused to the catalytic domain of human dual-specificity phosphatase 1 in complex with DARPins are reported.

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Unraveling the long-pursued Au144 structure by x-ray crystallography

Science Advances ◽

10.1126/sciadv.aat7259 ◽

2018 ◽

Vol 4 (10) ◽

pp. eaat7259 ◽

Cited By ~ 112

Author(s):

Nan Yan ◽

Nan Xia ◽

Lingwen Liao ◽

Min Zhu ◽

Fengming Jin ◽

...

Keyword(s):

Metal Nanoparticles ◽

Weak Interactions ◽

Gold Nanoclusters ◽

Atomic Level ◽

High Quality ◽

X Ray ◽

X Ray Crystallography ◽

Quantum Size ◽

First Time

The transition from nanocluster to nanocrystal is a central issue in nanoscience. The atomic structure determination of metal nanoparticles in the transition size range is challenging and particularly important in understanding the quantum size effect at the atomic level. On the basis of the rationale that the intra- and interparticle weak interactions play critical roles in growing high-quality single crystals of metal nanoparticles, we have reproducibly obtained ideal crystals of Au144(SR)60 and successfully solved its structure by x-ray crystallography (XRC); this structure was theoretically predicted a decade ago and has long been pursued experimentally but without success until now. Here, XRC reveals an interesting Au12 hollow icosahedron in thiolated gold nanoclusters for the first time. The Au–Au bond length, close to that of bulk gold, shows better thermal extensibility than the other Au–Au bond lengths in Au144(SR)60, providing an atomic-level perspective because metal generally shows better thermal extensibility than nonmetal materials. Thus, our work not only reveals the mysterious, long experimentally pursued structure of a transition-sized nanoparticle but also has important implications for the growth of high-quality, single-crystal nanoparticles, as well as for the understanding of the thermal extensibility of metals from the perspective of chemical bonding.

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Crystallization of the 10-23 DNA enzyme using a combinatorial screen of paired oligonucleotides

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444999010550 ◽

1999 ◽

Vol 55 (11) ◽

pp. 1885-1892 ◽

Cited By ~ 17

Author(s):

Jacek Nowakowski ◽

Peter J. Shim ◽

Gerald F. Joyce ◽

C. David Stout

Keyword(s):

Crystal Formation ◽

High Quality ◽

Acid Complex ◽

Crystal Forms ◽

X Ray ◽

X Ray Crystallography ◽

Crystallization Solution ◽

Varying Length ◽

Standard Set

One of the most difficult steps in the X-ray crystallography of nucleic acids is obtaining crystals that diffract to high resolution. The choice of the nucleotide sequence has proven to be more important in producing high-quality crystals than the composition of the crystallization solution. This manuscript describes a systematic procedure for identifying the optimal sizes of a multi-stranded nucleic acid complex which provide high-quality crystals. This approach was used to crystallize the in vitro evolved 10-23 DNA enzyme complexed with its RNA substrate. In less than two months, 81 different enzyme–substrate complexes were generated by combinatorial mixing and annealing of complementary oligonucleotides which differed in length, resulting in duplexes of varying length, with or without nucleotide overhangs. Each of these complexes was screened against a standard set of 48 crystallization conditions and evaluated for crystal formation. The screen resulted in over 40 crystal forms, the best of which diffracted to 2.8 Å resolution when exposed to a synchrotron X-ray source.

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High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phl p 2 As Revealed by X-Ray Crystallography

The Journal of Immunology ◽

10.4049/jimmunol.0803018 ◽

2009 ◽

Vol 182 (4) ◽

pp. 2141-2151 ◽

Cited By ~ 83

Author(s):

Sivaraman Padavattan ◽

Sabine Flicker ◽

Tilman Schirmer ◽

Christoph Madritsch ◽

Stefanie Randow ◽

...

Keyword(s):

Conformational Epitope ◽

X Ray ◽

X Ray Crystallography ◽

High Affinity

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Crystal structure of glycosyltrehalose synthase fromSulfolobus shibataeDSM5389

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1801453x ◽

2018 ◽

Vol 74 (11) ◽

pp. 741-746

Author(s):

Nobuo Okazaki ◽

Michael Blaber ◽

Ryota Kuroki ◽

Taro Tamada

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Structural Basis ◽

Hyperthermophilic Archaeon ◽

X Ray ◽

X Ray Crystallography ◽

Sulfolobus Shibatae ◽

Glucosidic Bond

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeonSulfolobus shibataeDSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the (β/α)8-barrel fold that is conserved as the catalytic domain in the α-amylase family. Three invariant catalytic carboxylic amino acids in the α-amylase family are also found in GTSase at positions Asp241, Glu269 and Asp460 in the catalytic domain. The shape of the catalytic cavity and the pocket size at the bottom of the cavity correspond to the intramolecular transglycosylation mechanism proposed from previous enzymatic studies.

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Inhibitors of the catalytic domain of mitochondrial ATP synthase

Biochemical Society Transactions ◽

10.1042/bst0340989 ◽

2006 ◽

Vol 34 (5) ◽

pp. 989-992 ◽

Cited By ~ 17

Author(s):

J.R. Gledhill ◽

J.E. Walker

Keyword(s):

Atp Synthase ◽

Binding Sites ◽

Catalytic Domain ◽

Structural Studies ◽

Inhibitor Protein ◽

X Ray ◽

F1 Atpase ◽

X Ray Crystallography ◽

Mitochondrial Atp Synthase ◽

Natural Inhibitor

An understanding of the mechanism of ATP synthase requires an explanation of how inhibitors act. The catalytic F1-ATPase domain of the enzyme has been studied extensively by X-ray crystallography in a variety of inhibited states. Four independent inhibitory sites have been identified by high-resolution structural studies. They are the catalytic site, and the binding sites for the antibiotics aurovertin and efrapeptin and for the natural inhibitor protein, IF1.

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Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

IUCrJ ◽

10.1107/s2052252514014900 ◽

2014 ◽

Vol 1 (5) ◽

pp. 305-317 ◽

Cited By ~ 5

Author(s):

Ho-Hsien Lee ◽

Irene Cherni ◽

HongQi Yu ◽

Raimund Fromme ◽

Jeffrey D. Doran ◽

...

Keyword(s):

Fusion Protein ◽

Structural Information ◽

Proximal Region ◽

Two Dimensions ◽

Three Dimensions ◽

Mucosal Vaccine ◽

X Ray ◽

X Ray Crystallography ◽

Third Dimension ◽

Hiv 1

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein ofHuman immunodeficiency virus 1(HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed inEscherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

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A New, Simple, High-Affinity Glycosidase Inhibitor: Analysis of Binding through X-ray Crystallography, Mutagenesis, and Kinetic Analysis

Journal of the American Chemical Society ◽

10.1021/ja0002870 ◽

2000 ◽

Vol 122 (17) ◽

pp. 4229-4230 ◽

Cited By ~ 33

Author(s):

Spencer J. Williams ◽

Valerie Notenboom ◽

Jacqueline Wicki ◽

David R. Rose ◽

Stephen G. Withers

Keyword(s):

Kinetic Analysis ◽

X Ray ◽

X Ray Crystallography ◽

High Affinity ◽

Inhibitor Analysis ◽

Glycosidase Inhibitor

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Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21008967 ◽

2021 ◽

Vol 77 (10) ◽

Author(s):

Takeshi Murakawa ◽

Mamoru Suzuki ◽

Toshi Arima ◽

Michihiro Sugahara ◽

Tomoyuki Tanaka ◽

...

Keyword(s):

Free Electron ◽

Amine Oxidase ◽

Protein Structures ◽

Copper Amine Oxidase ◽

High Quality ◽

Uniform Size ◽

Widespread Application ◽

X Ray ◽

X Ray Crystallography ◽

Copper Amine

Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5–15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3–5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.

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Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01026-10 ◽

2010 ◽

Vol 76 (19) ◽

pp. 6351-6359 ◽

Cited By ~ 7

Author(s):

Toshihiko Kitajima ◽

Yasunori Chiba ◽

Yoshifumi Jigami

Keyword(s):

Saccharomyces Cerevisiae ◽

Methylotrophic Yeast ◽

Culture Conditions ◽

Crystallographic Analysis ◽

Gene Encoding ◽

X Ray ◽

X Ray Crystallography ◽

High Affinity ◽

Standard Culture ◽

Resistant Mutants

ABSTRACT The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

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