Generation, representation and flow of phase information in structure determination: recent developments in and around <i>SHARP</i> 2.0

Bricogne, G.; Vonrhein, Clemens; Flensburg, Claus; Schiltz, M.; Paciorek, W. A.

doi:10.1107/s0907444903017694

Cited by 603 publications

(505 citation statements)

References 14 publications

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“…The crystals diffracted X-rays to 2.3 Å resolution and each asymmetric unit contains one ICMT-monobody complex. Initial experimental phases (35 – 4 Å) for the ICMT-monobody complex (space group P2 1 2 1 2 1 ) were determined using the SIRAS phasing method in SHARP and improved using solvent flattening 33 . Five Se-Met sites were located and correspond to the five methionine residues of ICMT.…”

Section: Methodsmentioning

confidence: 99%

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Diver

Pedi

Koide

et al. 2018

Nature

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The maturation of Ras GTPases, and ~200 other cellular CaaX proteins, involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CaaX motif, proteolytic cleavage of the aaX residues, and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum (ER)2. ICMT is the only cellular enzyme known to methylate prenylcysteine substrates; methylation is important for their biological functions, including the membrane localisations and subsequent activities of Ras1, prelamin A3, and Rab4. ICMT inhibition has potential for combating progeria3 and cancer5–8. Here we present an X-ray structure of ICMT, at 2.3 Å resolution, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for its cytosolic methyl donor, S-adenosyl-L-methionine (AdoMet), and for prenylcysteine substrates, which are associated with the ER membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localisations together in a membrane environment – a relatively uncharacterized, but defining feature of many integral membrane enzymes.

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Section: Methodsmentioning

confidence: 99%

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Diver

Pedi

Koide

et al. 2018

Nature

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“…The six selenium sites were found by using SHELXD (11). Phasing with the program SHARP (12) produced an electron density map of good quality. The program WARP (13) was used to automatically build 470 of 542 residues.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of a Baeyer–Villiger monooxygenase

Malito

Alfieri²,

Fraaije³

et al. 2004

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

275

305

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Flavin-containing Baeyer-Villiger monooxygenases employ NADPH and molecular oxygen to catalyze the insertion of an oxygen atom into a carbon-carbon bond of a carbonylic substrate. These enzymes can potentially be exploited in a variety of biocatalytic applications given the wide use of Baeyer-Villiger reactions in synthetic organic chemistry. The catalytic activity of these enzymes involves the formation of two crucial intermediates: a flavin peroxide generated by the reaction of the reduced flavin with molecular oxygen and the ''Criegee'' intermediate resulting from the attack of the flavin peroxide onto the substrate that is being oxygenated. The crystal structure of phenylacetone monooxygenase, a Baeyer-Villiger monooxygenase from the thermophilic bacterium Thermobifida fusca, exhibits a two-domain architecture resembling that of the disulfide oxidoreductases. The active site is located in a cleft at the domain interface. An arginine residue lays above the flavin ring in a position suited to stabilize the negatively charged flavin-peroxide and Criegee intermediates. This amino acid residue is predicted to exist in two positions; the ''IN'' position found in the crystal structure and an ''OUT'' position that allows NADPH to approach the flavin to reduce the cofactor. Domain rotations are proposed to bring about the conformational changes involved in catalysis. The structural studies highlight the functional complexity of this class of flavoenzymes, which coordinate the binding of three substrates (molecular oxygen, NADPH, and phenylacetone) in proximity of the flavin cofactor with formation of two distinct catalytic intermediates.flavoenzyme ͉ mechanism of catalysis ͉ biocatalysis ͉ Baeyer-Villiger reaction ͉ crystallography A t the end of the 19th century, Baeyer and Villiger (1) discovered that cyclic ketones react with oxidants, such as peroxymonosulfuric acid, to yield lactones. The mechanism of the Baeyer-Villiger reaction involves a nucleophilic attack of a peroxide to the ketone reagent to generate the so-called ''Criegee'' intermediate ( Fig. 1), which is followed by an intramolecular rearrangement that leads to the migration of an alkyl group to an oxygen atom, generating the lactone product. Baeyer-Villiger reactions are of enormous value in synthetic organic chemistry, and the number of their applications is countless.Several microorganisms produce enzymes capable to catalyze Baeyer-Villiger reactions. These proteins are extensively studied for their exploitation in biocatalytic applications (2, 3). This interest follows the problems related to the toxicity and instability of the oxidizing reactants that are currently being used in chemical processes. In addition, enzymatic reactions exhibit a superior degree of enantio-and regioselectivity. Baeyer-Villiger monooxygenases are classified depending on the nature of their flavin cofactor (4). The type I enzymes are the most extensively investigated (5). They are FAD-dependent proteins that use NADPH and molecular oxygen to insert an oxygen atom into th...

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“…The phase problem was solved by using bovine TC and single-wavelength anomalous dispersion data to 2.8-Å resolution collected on the trigonal crystal form at the Co K-edge with 16-fold multiplicity. The Co coordinates were found by using SHELXD (42), and initial phases were calculated with SHARP (43). A partial protein model was obtained by interpretation of solvent-flattened electrondensity maps by using the graphics program O (44) and automated procedures in ARP͞WARP (45).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for mammalian vitamin B ₁₂ transport by transcobalamin

Wuerges

Garau²,

Geremia³

et al. 2006

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

166

216

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Cobalamin (Cbl, vitamin B12) serves for two essential cofactors in mammals. The pathway for its intestinal absorption, plasma transport, and cellular uptake uses cell surface receptors and three Cbl-transporting proteins, haptocorrin, intrinsic factor, and transcobalamin (TC). We present the structure determination of a member of the mammalian Cbl-transporter family. The crystal structures of recombinant human and bovine holo-TCs reveal a two-domain architecture, with an N-terminal ␣6-␣6 barrel and a smaller C-terminal domain. One Cbl molecule in base-on conformation is buried inside the domain interface. Structural data combined with previous binding assays indicate a domain motion in the first step of Cbl binding. In a second step, the weakly coordinated ligand H 2O at the upper axial side of added H2O-Cbl is displaced by a histidine residue of the ␣6-␣6 barrel. Analysis of amino acid conservation on TC's surface in orthologous proteins suggests the location of the TC-receptor-recognition site in an extended region on the ␣6-␣6 barrel. The TC structure allows for the mapping of sites of amino acid variation due to polymorphisms of the human TC gene. Structural information is used to predict the overall fold of haptocorrin and intrinsic factor and permits a rational approach to the design of new Cbl-based bioconjugates for diagnostic or therapeutic drug delivery.cobalamin ͉ crystal structure ͉ P259R polymorphism ͉ transport protein

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Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0

Cited by 603 publications

References 14 publications

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Crystal structure of a Baeyer–Villiger monooxygenase

Structural basis for mammalian vitamin B ₁₂ transport by transcobalamin

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Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0

Cited by 603 publications

References 14 publications

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Crystal structure of a Baeyer–Villiger monooxygenase

Structural basis for mammalian vitamin B 12 transport by transcobalamin

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Product

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Structural basis for mammalian vitamin B ₁₂ transport by transcobalamin