Post-crystallization treatments for improving diffraction quality of protein crystals

Heras, Begoña; Martin, Jennifer L.

doi:10.1107/s0907444905019451

Cited by 199 publications

(192 citation statements)

References 45 publications

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“…The crystals were cryoprotected by slow transfer to artificial mother liquor supplemented with 30% PEG 400. The diffraction qualities of the crystals were improved significantly by extensive trials of dehydration (32). The crystals were dehydrated by stepwise transfer to a mother liquor containing 25% PEG 3350, 0.2 M di-ammonium citrate hexahydrate, and 0.2 M sodium thiocyanate and were equilibrated over the same mother liquor for 5 d before cryoprotection.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration

Lee

Greenlee

Amick

et al. 2015

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

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ErbB3 (HER3) is a member of the EGF receptor (EGFR) family of receptor tyrosine kinases, which, unlike the other three family members, contains a pseudo kinase in place of a tyrosine kinase domain. In cancer, ErbB3 activation is driven by a ligand-dependent mechanism through the formation of heterodimers with EGFR, ErbB2, or ErbB4 or via a ligand-independent process through heterodimerization with ErbB2 overexpressed in breast tumors or other cancers. Here we describe the crystal structure of the Fab fragment of an antagonistic monoclonal antibody KTN3379, currently in clinical development in human cancer patients, in complex with the ErbB3 extracellular domain. The structure reveals a unique allosteric mechanism for inhibition of ligand-dependent or ligand-independent ErbB3-driven cancers by binding to an epitope that locks ErbB3 in an inactive conformation. Given the similarities in the mechanism of ErbB receptor family activation, these findings could facilitate structure-based design of antibodies that inhibit EGFR and ErbB4 by an allosteric mechanism.cancer | surface receptor | cell signaling | therapeutic antibodies | crystal structure T he EGF receptor (EGFR)/ErbB family of receptor tyrosine kinases (RTKs) participates in a multitude of roles during embryonic development and in adult homeostasis. In healthy tissues, ErbB signal transduction is initiated through ligandinduced homo-or heterodimerization of receptor extracellular domains leading to the stimulation of tyrosine kinase activity and autophosphorylation of several tyrosine residues in the cytoplasmic domain followed by recruitment and activation of multiple downstream signaling pathways (1). In contrast, unregulated ErbB signaling through activating mutations, receptor overexpression, or aberrant autocrine ligand signaling loops can lead to cellular transformation and tumorigenesis (2). Thus, members of the ErbB receptor family (in particular EGFR and ErbB2) have become well-validated targets for the development of anticancer therapeutics, resulting in a number of Food and Drug Administration-approved and marketed monoclonal antibodies and small-molecule tyrosine kinase inhibitors used for treatment of different cancers (3).The activity of ErbB3 (also designated HER3) is normally regulated by the neuregulin (NRG) family of growth factors (4), but, unlike other members of the family, ErbB3 functions as an obligate heterodimer with other ErbB receptors because its cytoplasmic domain contains a pseudo kinase in place of a tyrosine kinase domain (5, 6). ErbB3 therefore takes part in heterodimer formation through ligand binding, whereas its coreceptor (most often ErbB2) provides enzymatic activity to phosphorylate multiple tyrosine residues located primarily in the ErbB3 C-terminal tail. The role of ErbB3 in cancer has been fully appreciated only within the last decade and has prompted the development of several monoclonal antibodies geared at inhibiting its action in solid tumors. ErbB3 phosphorylation and subsequent signaling have been associated wi...

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Section: Methodsmentioning

confidence: 99%

Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration

Lee

Greenlee

Amick

et al. 2015

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

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“…GraFix is thus unlikely to cause artifacts that can be attributed to chemical fixation at intermediate resolution. We expect that glutaraldehyde or formaldehyde treatment might allow higher-resolution structure determination as it has been shown that high-resolution X-ray structures can be obtained in the presence of chemical fixation reagents 12,13 . We tested GraFix on various fragile complexes with low cellular copy number such as spliceosomes (Fig.…”

mentioning

confidence: 99%

GraFix: sample preparation for single-particle electron cryomicroscopy

et al. 2007

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“…9). The effect of dehydration on WbdD (an improvement in diffraction resolution from 8 to 2.2 Å ) is very pronounced when compared with other examples (Heras & Martin, 2005). However, a facile and easily implementable protocol may extend the utility of dehydration.…”

Section: Effect Of Dehydration On the Structurementioning

confidence: 97%

“…However, a facile and easily implementable protocol may extend the utility of dehydration. The procedure described here combines several previously published ideas (Kiefersauer et al, 2000;Heras & Martin, 2005) into an easy-to-use, low-cost and convenient workflow. It is not anticipated that this will necessarily lead to more success than other approaches, but it is straightforward and amenable to high throughput.…”

Section: Effect Of Dehydration On the Structurementioning

confidence: 99%

Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase fromEscherichia coliO9a

Hagelueken

Huang

Harlos

et al. 2012

Acta Crystallogr D Biol Cryst

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WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å ) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å ) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.

show abstract

Post-crystallization treatments for improving diffraction quality of protein crystals

Cited by 199 publications

References 45 publications

Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration

Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration

GraFix: sample preparation for single-particle electron cryomicroscopy

Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase fromEscherichia coliO9a

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