Combining experimental data for structure determination of flexible multimeric macromolecules by molecular replacement

Trapani, Stefano; Abergel, Chantal; Gutsche, Irina; Horcajada, Cristina; Fita, Ignacio; Navaza, Jorge

doi:10.1107/s0907444906005361

Cited by 9 publications

(8 citation statements)

References 20 publications

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“…Electron microscopy - Activated ECAM at a concentration of 0.05 mg/mL was first applied to the clean side of carbon on mica (carbon/mica interface) and negatively stained with 1% (w/v) sodium silico tungstate [40]. A grid was placed on top of the carbon film, and subsequently air-dried.…”

Section: Methodsmentioning

confidence: 99%

Conformational States of a Bacterial α2-Macroglobulin Resemble Those of Human Complement C3

et al. 2012

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α2 macroglobulins (α2Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α2Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α2M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α2M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.

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Section: Methodsmentioning

confidence: 99%

Conformational States of a Bacterial α2-Macroglobulin Resemble Those of Human Complement C3

et al. 2012

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“…The structure of this purified domain was next solved by molecular replacement despite the low overall sequence similarity (20-25%) detected with known protein kinases, by taking advantage of a structure-ensemble computed from 10 distinct templates, as a search model to increased signal-to-noise-ratio (Trapani et al, 2006). The crystal structure was refined to a Rwork/Rfree of 19.0/24.3 at a 2.8 Å resolution with two molecules in the asymmetric unit ( Supplementary Table S1).…”

Section: Overall Structure and Topology Of Pragmin Kinase Domainmentioning

confidence: 99%

Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

Lecointre

Simon

Kerneur

et al. 2017

Preprint

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The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N-and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.All rights reserved. No reuse allowed without permission.

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“…Other simpler approaches consist of generating systematically perturbed models along one particular mode using, for example, the elNémo web server and then proceeding with the standard MR procedure into the cryo-EM map (Trapani et al, 2006) for each perturbed model.…”

Section: Nma and Cryo-em (Flexible Fitting)mentioning

confidence: 99%

Dealing with structural variability in molecular replacement and crystallographic refinement through normal-mode analysis

Delarue

2007

Acta Crystallogr D Biol Cryst

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Normal‐mode analysis (NMA) can be used to generate multiple structural variants of a given template model, thereby increasing the chance of finding the molecular‐replacement solution. Here, it is shown that it is also possible to directly refine the amplitudes of the normal modes against experimental data (X‐ray or cryo‐EM), generalizing rigid‐body refinement methods by adding just a few additional degrees of freedom that sample collective and large‐amplitude movements. It is also argued that the situation where several (conformations of) models are present simultaneously in the crystal can be studied with adjustable occupancies using techniques derived from statistical thermodynamics and already used in molecular modelling.

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Combining experimental data for structure determination of flexible multimeric macromolecules by molecular replacement

Cited by 9 publications

References 20 publications

Conformational States of a Bacterial α2-Macroglobulin Resemble Those of Human Complement C3

Conformational States of a Bacterial α2-Macroglobulin Resemble Those of Human Complement C3

Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

Dealing with structural variability in molecular replacement and crystallographic refinement through normal-mode analysis

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