Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1

Svensson, Olof; Gilski, Maciej; Nurizzo, Didier; Bowler, Matthew W.

doi:10.1107/s2059798318003728

Cited by 41 publications

(33 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…X-ray diffraction data were collected from a single point on the crystal using automatic protocols for the location and optimal centring of crystals 43 . The beam diameter was selected automatically to match the crystal volume of highest homogeneous quality and was therefore collimated to 30 µm, and strategy calculations accounted for flux and crystal volume in the parameter prediction for complete data sets 44 . Diffraction data were processed using MOSFLM 45 and AIMLESS 46 , the structure was solved using PHASER 47 with use of the crystal structures of the active state β 2 AR stabilized with nanobody Nb80 6 and wild-type thioredoxin (PDB codes 3P0G, 2H6X) as search models.…”

Section: Methodsmentioning

confidence: 99%

Molecular determinants of β-arrestin coupling to formoterol-bound β₁-adrenoceptor

Warne

Nehmé

et al. 2020

Preprint

View full text Add to dashboard Cite

36The β 1 -adrenoceptor (β 1 AR) is a G protein-coupled receptor (GPCR) 37 activated by the hormone noradrenaline, resulting in the coupling of the 38 heterotrimeric G protein G s 1 . G protein-mediated signalling is terminated by 39 phosphorylation of the receptor C-terminus and coupling of β-arrestin 1 (βarr1, 40 also known as arrestin-2), which displaces G s and induces signalling through the 41 MAP kinase pathway 2 . The ability of synthetic agonists to induce signalling 42 preferentially through either G proteins or arrestins (biased agonism) 3 is 43 important in drug development, as the therapeutic effect may arise from only 44 one signalling cascade, whilst the other pathway may mediate undesirable side 45 effects 4 . To understand the molecular basis for arrestin coupling, we determined 46 the electron cryo-microscopy (cryo-EM) structure of the β 1 AR-βarr1 complex in 47 lipid nanodiscs bound to the biased agonist formoterol 5 , and the crystal structure 48 of formoterol-bound β 1 AR coupled to the G protein mimetic nanobody Nb80 6 . 49 βarr1 couples to β 1 AR in a distinct manner to how G s couples to β 2 AR 7 , with the 50 finger loop of βarr1 occupying a narrower cleft on the intracellular surface 51 closer to transmembrane helix H7 than the C-terminal α5 helix of G s . The 52 conformation of the finger loop in βarr1 is different from that adopted by the 53 finger loop in visual arrestin when it couples to rhodopsin 8 , and its β-turn 54 configuration is reminiscent of the loop in Nb80 that inserts at the same position. 55 β 1 AR coupled to βarr1 showed significant differences in structure compared to 56 β 1 AR coupled to Nb80, including an inward movement of extracellular loop 3 57 (ECL3) and the cytoplasmic ends of H5 and H6. In the orthosteric binding site 58 there was also weakening of interactions between formoterol and the residues 59 Ser211 5.42 and Ser215 5.46 , and a reduction in affinity of formoterol for the β 1 AR-60 βarr1 complex compared to β 1 AR coupled to mini-G s . These differences provide 61 a foundation for the development of small molecules that could bias signalling in 62 the β-adrenoceptors. 63 64 65Ligand bias arises through differential activation of the G protein pathway 66 compared to the arrestin pathway and has been observed in ligands binding to many 67 different GPCRs such as the µ-opioid receptor 9 , the angiotensin receptor (AT 1 R) 10 68 10 th December 2019 3 and the β-adrenoceptors, β 1 AR and β 2 AR 5,11 . The ligands can show complex 69 pharmacology. For example, carvedilol is an inverse agonist of β 1 AR when G protein 70 activity is measured, but it is a weak agonist of the MAPK pathway activated by 71 βarr1 11 . In contrast formoterol is an agonist of both pathways, but stimulates the βarr1 72 pathway more than the G protein pathway 5 . Current theories favour the hypothesis 73 that GPCRs exist in an ensemble of conformations and that ligands preferentially 74 stabilise specific conformations 12 . This suggests that the conformation of a receptor 75 bound to a h...

show abstract

Section: Methodsmentioning

confidence: 99%

Molecular determinants of β-arrestin coupling to formoterol-bound β₁-adrenoceptor

Warne

Nehmé

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…X-ray data were collected, processed, and analyzed in a fully automated fashion at MASSIF-1 on beamline ID30A-1 at the European synchrotron radiation facility (ESRF). 8 The starting phases were obtained by molecular replacement using PHASER 9 with an available Mps1 kinase structure (PDB code: 3HMN 6 ) as the search model. The models were built and refined using iterative cycles of COOT, 9 REFMAC, 9 and PDB-REDO.…”

Section: Data Collection and Structure Refinementmentioning

confidence: 99%

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop

et al. 2019

View full text Add to dashboard Cite

Monopolar spindle 1 (Mps1) is a dual‐specificity protein kinase, orchestrating faithful chromosome segregation during mitosis. All reported structures of the Mps1 kinase adopt the hallmarks of an inactive conformation, which includes a mostly disordered activation loop. Here, we present a 2.4 Å resolution crystal structure of an “extended” version of the Mps1 kinase domain, which shows an ordered activation loop. However, the other structural characteristics of an active kinase are not present. Our structure shows that the Mps1 activation loop can fit to the ATP binding pocket and interferes with ATP, but less so with inhibitors binding, partly explain the potency of various Mps1 inhibitors.

show abstract

“…in determining the diameter of the beam that should be used to maximise the signal to noise ratio (Evans et al, 2011;Svensson et al, 2018). However, the gathering of volumetric data has many other potential uses, not least being able to correlate crystal size to quality for individual projects.…”

Section: The Size and Shape Of Protein Crystalsmentioning

confidence: 99%

A comparative anatomy of protein crystals: lessons from the automatic processing of 56,000 samples

Svensson

Gilski

Nurizzo

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

SynopsisThe automatic processing of over 56,000 crystals by the autonomous ESRF beamline MASSIF-1 has provided a data set of crystal characteristics and properties that allows many theoretical proposals and assumptions to be evaluated experimentally. AbstractThe fully automatic processing of crystals of macromolecules has presented a unique opportunity to gather information on the samples that is not usually recorded. This has proved invaluable in improving the sample location, characterisation and data collection algorithms. After operating for four years, MASSIF-1 has now processed over 56,000 samples, gathering information at each stage, from the volume of the crystal to the unit cell dimensions, space group, quality of the data collected and the reasoning behind the decisions made in data collection. This provides an unprecedented opportunity to analyse these data together, providing a detailed landscape of macromolecular crystals and intimate details of their contents and, importantly, how the two are related. The data show that mosaic spread is unrelated to the size or shape of crystals and demonstrate experimentally that diffraction intensities scale in proportion to crystal volume and molecular weight. It is also shown that crystal volume scales inversely with molecular weight.The results set the scene for the development of X-ray crystallography in a changing environment for structural biology.

show abstract

Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1

Abstract: Significant improvements to the sample-location, characterization and data-collection algorithms on the autonomous ESRF beamline MASSIF-1 are described. The workflows now include dynamic beam-diameter adjustment and multi-position and multi-crystal data collections.

Cited by 41 publications

References 55 publications

Molecular determinants of β-arrestin coupling to formoterol-bound β₁-adrenoceptor

Molecular determinants of β-arrestin coupling to formoterol-bound β₁-adrenoceptor

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop

A comparative anatomy of protein crystals: lessons from the automatic processing of 56,000 samples

Contact Info

Product

Resources

About

Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1

Abstract: Significant improvements to the sample-location, characterization and data-collection algorithms on the autonomous ESRF beamline MASSIF-1 are described. The workflows now include dynamic beam-diameter adjustment and multi-position and multi-crystal data collections.

Cited by 41 publications

References 55 publications

Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop

A comparative anatomy of protein crystals: lessons from the automatic processing of 56,000 samples

Contact Info

Product

Resources

About

Molecular determinants of β-arrestin coupling to formoterol-bound β₁-adrenoceptor

Molecular determinants of β-arrestin coupling to formoterol-bound β₁-adrenoceptor