Improved Structure Refinement Through Maximum Likelihood

Pannu, Navraj S.; Read, Randy J.

doi:10.1107/s0108767396004370

Cited by 346 publications

(278 citation statements)

References 16 publications

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“…Coordinates from the affinity-matured 28B4 were used as a model for the germline Fab-hapten structure, and this structure was used as a model for the unliganded germline Fab structure. Alternate rounds of model building in O (14) and refinement of the coordinates using CNS version 0.4 (15)(16)(17) were performed until minimization was complete. A total of 278 waters were added to the unliganded germline Fab structure, and no waters were added to the germline Fab-hapten structure.…”

Section: Reagents and Strainsmentioning

confidence: 99%

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

et al. 2001

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In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K d ) 25 µM) and 28B4 Fab (K d ) 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95 H Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. ( ) Science 279, 1929. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.The evolution of binding affinity in the immune system involves combinatorial and mutational processes, which in many aspects parallel those that occur in the natural evolution of enzymes. Consequently, the characterization of the affinity mat...

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Section: Reagents and Strainsmentioning

confidence: 99%

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

et al. 2001

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“…The structure of Stx had been determined at lower resolution (2.5 Å) (27) than that of Stx2 (1.77 Å), so we hypothesized that the model for Stx could be improved by using information from the structure of Stx2. As well, Stx had been refined prior to the use of the maximum likelihood target in refinement (28). For these reasons, the Stx model was refined for several additional cycles using the original data set (5).…”

Section: Methodsmentioning

confidence: 99%

Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7

Fraser

Fujinaga

Cherney

et al. 2004

Journal of Biological Chemistry

266

280

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Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1. These Stx-producing E. coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E. coli produce Stx2 than if they only produce Stx1. To investigate the differences among the Stxs, which are all AB 5 toxins, the crystal structure of Stx2 from E. coli O157:H7 was determined at 1.8-Å resolution and compared with the known structure of Stx. Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate. Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-subunit. Of the three types of receptor-binding sites in the B-pentamer, one has a different conformation in Stx2 than in Stx, and the carboxyl terminus of the A-subunit binds at another. Any of these structural differences might result in different mechanisms of action of the two toxins and the development of hemolytic uremic syndrome upon exposure to Stx2.

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“…The ML CV approach proved to be more convergent than the LSQ function (Pannu & Read, 1996). This work suggests that one can achieve even better convergence towards the true structure by the use of the ML FK approach with larger work sets.…”

Section: Discussionmentioning

confidence: 88%

“…The calculated structure factors include bulk-solvent correction, whereas the F obs are modified by the overall anisotropic B correction. The notation I o represents the modified Bessel functions and the parameters and define the expected phase errors as described in the literature (Lunin & Urzhumtsev, 1984;Read, 1986;Lunin & Skovoroda, 1995;Pannu & Read, 1996). The MAIN algorithm is an implementation of the formulation described by Lunin & Skovoroda (1995).…”

Section: Refinement Protocolmentioning

confidence: 99%

“…Use of the partial free data concept (Lunin & Skovoroda, 1995) showed that the refinement was successful. As a consequence, ML refinement has been widely adopted by the crystallographic community (Pannu & Read, 1996;Bricogne & Irwin, 1996;Murshudov et al, 1997;Adams et al, 1997;Pannu et al, 1998). Today, the free, model-unbiased fraction of data typically consists of 5% of the diffraction data.…”

Section: Introductionmentioning

confidence: 99%

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Free kick instead of cross-validation in maximum-likelihood refinement of macromolecular crystal structures

Pražnikar

Turk

2014

Acta Cryst D Biol Crystallogr

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The refinement of a molecular model is a computational procedure by which the atomic model is fitted to the diffraction data. The commonly used target in the refinement of macromolecular structures is the maximum-likelihood (ML) function, which relies on the assessment of model errors. The current ML functions rely on cross-validation. They utilize phase-error estimates that are calculated from a small fraction of diffraction data, called the test set, that are not used to fit the model. An approach has been developed that uses the work set to calculate the phase-error estimates in the ML refinement from simulating the model errors via the random displacement of atomic coordinates. It is called ML free-kick refinement as it uses the ML formulation of the target function and is based on the idea of freeing the model from the model bias imposed by the chemical energy restraints used in refinement. This approach for the calculation of error estimates is superior to the cross-validation approach: it reduces the phase error and increases the accuracy of molecular models, is more robust, provides clearer maps and may use a smaller portion of data for the test set for the calculation of R free or may leave it out completely.

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Improved Structure Refinement Through Maximum Likelihood

Cited by 346 publications

References 16 publications

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7

Free kick instead of cross-validation in maximum-likelihood refinement of macromolecular crystal structures

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