Difference Fourier refinement of the structure of DIP-trypsin at 1.5 Å with a minicomputer technique

Chambers, J. L.; Stroud, R. M.

doi:10.1107/s0567740877007146

Cited by 87 publications

(64 citation statements)

References 20 publications

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“…In macromolecular crystallography, the number of atoms involved often prohibits completely unrestrained refinement, so restraints and constraints are applied. The number of structure analyses where restraints have been removed is few, though these structures, like trypsin when refined by difference Fourier methods and without constraints (Chambers & Stroud, 1977), offer a rich source of data. These data ultimately become built into useful restraints and con- Also indicated is the best-fit line to the data points at each B factor.…”

Section: Correlation Of Errors Wirh Measures Of Model Qualitymentioning

confidence: 99%

“…Such variances can be estimated directly from the gradient and curvature in electron density maps (Chambers & Stroud, 1977), however, these are not readily restored from information deposited in the Protein Data Bank. Because various constraints toward ideality of geometry may be applied during the crystallographic analysis, positional deviations may be further reduced from these values as they would be determined for unconstrained atoms.…”

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confidence: 99%

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Significance of structural changes in proteins: Expected errors in refined protein structures

Stroud

Fauman

1995

Protein Science

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A quantitative expression key to evaluating significant structural differences or induced shifts between any two protein structures is derived. Because crystallography leads to reports of a single (or sometimes dual) position for each atom, the significance of any structural change based on comparison of two structures depends critically on knowing the expected precision of each median atomic position reported, and on extracting it for each atom, from the information provided in the Protein Data Bank and in the publication. The differences between structures of protein molecules that should be identical, and that are normally distributed, indicating that they are not affected by crystal contacts, were analyzed with respect to many potential indicators of structure precision, so as to extract, essentially by "machine learning" principles, a generally applicable expression involving the highest correlates. Eighteen refined crystal structures from the Protein Data Bank, in which there are multiple molecules in the crystallographic asymmetric unit, were selected and compared. The thermal E factor, the connectivity of the atom, and the ratio of the number of reflections to the number of atoms used in refinement correlate best with the magnitude of the positional differences between regions of the structures that otherwise would be expected to be the same. These results are embodied in a six-parameter equation that can be applied to any crystallographically refined structure to estimate the expected uncertainty in position of each atom. Structure change in a macromolecule can thus be referenced to the expected uncertainty in atomic position as reflected in the variance between otherwise identical structures with the observed values of correlated parameters.

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Section: Correlation Of Errors Wirh Measures Of Model Qualitymentioning

confidence: 99%

mentioning

confidence: 99%

Significance of structural changes in proteins: Expected errors in refined protein structures

Stroud

Fauman

1995

Protein Science

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“…The rotation function yielded a clear solution, which gave an unambiguous translation solution. Difference Fourier refinement (22) and manual rebuilding using CHAIN (23) were interspersed with positional, B factor, and simulated annealing protocols in CNS to define the structure.…”

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confidence: 99%

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding

Chen

Krucinski

Miercke

et al. 2000

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

385

445

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“…Structures were solved in difference Fourier syntheses initially between the measured structure factor amplitudes (F o ) for the mutant complex and the measured structure factor amplitudes (F o ) for the wildtype enzyme (25) with reference to the structure of the L. casei TS-dUMP complex (5). Structures were refined in cycles by fitting to electron density maps calculated using either coeffecients (2|F o | -|F c |), or (|F o | -|F c |) and phases calculated from the coordinates, using the density fitting program Chain (26) (Figure 3).…”

Section: Novel Methylase By Protein Engineeringmentioning

confidence: 99%

A Novel dCMP Methylase by Engineering Thymidylate Synthase

et al. 1997

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X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH 2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 Å further than in the wild-type TS-dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH 2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 10 11 change in specificity for methylation of dCMP Versus dUMP. The structures of TS H199A/ N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) by 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 -folate) to form deoxythymidine-5′-monophosphate (dTMP) and 7,8-dihydrofolate (H 2 folate). Comparison of sequences of TS from ∼30 different organisms reveals that it is one of the most highly conserved enzymes (1, 2). A large number of three-dimensional structures of free and ligand-bound TSs and TS mutants have been determined and provide a structural understanding of substrate recognition, stereochemical aspects of the reaction pathway (3), and roles of specific residues in the reaction chemistry (4).Asn 229 is invariant in all TS's sequenced to date, and forms a cyclic hydrogen bond network with the 4-O and 3-NH of the substrate dUMP (Figure 2a) (5-7). Nevertheless, it can be replaced by numerous other residues, and the resultant mutants retain TS activity and also gain the capability to bind 2′-deoxycytidine 5′-monophosphate (dCMP) tightly (8,9). Moreover, alteration of Asn 229 to aspartate provides an enzyme which methylates dCMP more efficiently than dUMP, although the catalytic efficiency is still significantly lower than is the wild-type TS for its natural substrate dUMP (10, 11).

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Difference Fourier refinement of the structure of DIP-trypsin at 1.5 Å with a minicomputer technique

Cited by 87 publications

References 20 publications

Significance of structural changes in proteins: Expected errors in refined protein structures

Significance of structural changes in proteins: Expected errors in refined protein structures

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding

A Novel dCMP Methylase by Engineering Thymidylate Synthase

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