Comparative Studies of Protein Crystallization by Vapour-Diffusion and Microbatch Techniques

Chayen, Naomi E.

doi:10.1107/s0907444997005374

Cited by 105 publications

(95 citation statements)

References 42 publications

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“…Oligonucleotides were mixed in equimolar ratios in 100 mM Tris (pH 7.5), 40 mM MgCl 2 and annealed in a thermal cycler by denaturation at 94°C, followed by slow cooling to 4°C. The annealed DNA was then mixed in a 4:1 molar ratio with hPol μ Δ2 (10.7 mg/mL) and incubated on ice at 4°C for 1 h. Crystals of the binary complex were grown at room temperature by mixing 1 μL protein-DNA complex with 1 μL mother liquor [100 mM Tris, pH 8.5, 0.2 M MgCl 2 , 20% (vol/vol) PEG8000], using the sitting-drop vapor-diffusion technique (29). Crystals were transferred to a cryoprotectant solution containing 0.1 M Tris (pH 8.5), 50 mM NaCl, 0.2 M MgCl 2 , 20% (vol/vol) PEG8000, 5% (vol/vol) glycerol, and 10% (vol/vol) ethylene glycol.…”

mentioning

confidence: 99%

Creative template-dependent synthesis by human polymerase mu

Moon

Gosavi

Kunkel

et al. 2015

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

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Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol λ and Pol μ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol λ and Pol μ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol μ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol λ, Pol μ "skips" the first available template nucleotide, instead using the template base at the 5′ end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3′-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ.DNA polymerase mu | DNA polymerase lambda | DNA repair | nonhomologous end joining D NA double-strand breaks (DSBs) result from exposure to endogenous and exogenous factors including reactive oxygen species, physical or mechanical stress, ionizing radiation, or activities of nuclear enzymes on DNA (1). Another source of DSBs is programmed DNA breakage during meiotic or mitotic recombination, immunoglobin gene rearrangement in V(D)J, and class-switch recombination (1, 2). Because of the largely random nature of accidental double-strand breakage, the broken ends can have widely varying sequences, structures, or modifications. Nonhomologous end joining (NHEJ) is the predominant form of DSB repair in higher eukaryotes, and requires a certain degree of flexibility from the many factors involved in this complex repair process.DSB substrates lacking microhomology at the break site are unsuitable for immediate rejoining by ligase IV, and a polymerase is usually required to fill the gaps to generate ends that can be efficiently ligated (3). In higher eukaryotes, this role is performed by family X polymerase λ (Pol λ) and polymerase μ (Pol μ) (4-6). Pol λ has a strong preference for substrates with complementary template-strand pairing opposite the primer terminus. Pol μ can also use such complementary DSB substrates, but is uniquely active in template-dependent synthesis on DSBs entirely lacking complementarity, where the primer terminus is unpaired (7).Given partial overlap in substrate use in vitro by Pol μ and Pol λ, how does the NHEJ machinery select which enzyme to repair specific substrates with the highest fidelity? Recent work by Pryor et al. (8) (companion article in this issue) has demonstrated a strong preference for Pol λ over Pol μ in repairing the majority of complementary DSBs. This is advantageous, because Pol λ displays higher fidelity of synthesis (9, 10). However, Pol λ cannot use noncomplementary DSB substrates with unpaired primer termini. Pol μ is the only known polymerase that can use noncomplementary substrates, such that in Pol μ knockout cells, these ends are larg...

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mentioning

confidence: 99%

Creative template-dependent synthesis by human polymerase mu

Moon

Gosavi

Kunkel

et al. 2015

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

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“…Use optimization experiments also to improve reproducibility. 16,40,91 ; (d) crystallization by dialysis 7,92 ; (e) crystallization in gels [93][94][95] ); (x) protein purity, homogeneity and monodispersity.…”

Section: Optimization Of Crystallization Conditions-chemical and Physmentioning

confidence: 99%

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

2007

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The preparation of protein single crystals represents one of the major obstacles in obtaining the detailed 3D structure of a biological macromolecule. The complete automation of the crystallization procedures requires large investments in terms of money and labor, which are available only to large dedicated infrastructures and is mostly suited for genomic-scale projects. On the other hand, many research projects from departmental laboratories are devoted to the study of few specific proteins. Here, we try to provide a series of protocols for the crystallization of soluble proteins, especially the difficult ones, tailored for small-scale research groups. An estimate of the time needed to complete each of the steps described can be found at the end of each section

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“…42 The MBP-2OST protein crystals were obtained at 4 C using the sitting-drop vapor diffusion method. 42,83 The published structure of MBP (PDB ID code 1HSJ) was used to solve the phase problem via molecular replacement with the program MOLREP from the CCP4 Suite. 84 A single molecule of MBP was found within the asymmetric unit in this crystal form.…”

Section: Case Study #1: 2ost From Gallus Gallusmentioning

confidence: 99%

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

et al. 2010

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Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

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Comparative Studies of Protein Crystallization by Vapour-Diffusion and Microbatch Techniques

Cited by 105 publications

References 42 publications

Creative template-dependent synthesis by human polymerase mu

Creative template-dependent synthesis by human polymerase mu

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

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