<i>iMOSFLM</i>: a new graphical interface for diffraction-image processing with<i>MOSFLM</i>

Battye, T Geoff G; Kontogiannis, Luke; Johnson, Owen; Powell, Harold R.; Leslie, Andrew G. W.

doi:10.1107/s0907444910048675

Cited by 2,780 publications

(2,224 citation statements)

References 14 publications

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“…Of these frames, ~6.5% contained potential crystal hits as identified using Cheetah 27 (more than 15 Bragg peaks of minimum 2 pixels in size and a signal to noise ratio better than 7 after local background subtraction). Of the 91,626 potential crystal hits, 57,573 diffraction patterns could be auto-indexed by CrystFEL 28 (indexing rate of 63%) using a combination of MOSFLM 29 , asdf 28 and DirAx 30 . Reflections from different crystals in random orientations were merged using a Monte Carlo integration of each reflection by CrystFEL 28 .…”

Section: Methodsmentioning

confidence: 99%

Structure of the full-length glucagon class B G-protein-coupled receptor

Zhang

Qiao

Yang

et al. 2017

Nature

189

172

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The human glucagon receptor (GCGR) belongs to the class B G protein-coupled receptor (GPCR) family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both extracellular domain (ECD) and transmembrane domain (TMD) in an inactive conformation. The two domains are connected by a 12-residue segment termed the ‘stalk’, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of GCGR-TMD. The first extracellular loop (ECL1) exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen/deuterium exchange, disulfide cross-linking and molecular dynamics studies suggest that the stalk and ECL1 play critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding about the signaling mechanisms of class B GPCRs.

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Section: Methodsmentioning

confidence: 99%

Structure of the full-length glucagon class B G-protein-coupled receptor

Zhang

Qiao

Yang

et al. 2017

Nature

189

172

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“…Crystals were flash frozen into liquid nitrogen prior to data collection. Data were collected at beamline ID23‐1 at ERSF and processed with iMOSFLM (Battye et al ., 2011). Data collection statistics are summarized in Table 3.…”

Section: Methodsmentioning

confidence: 99%

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

Beek

Breton

Ferenbach

et al. 2015

Molecular Microbiology

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SummaryThe sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A S treptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium S almonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gac A in a S. mutans rml D knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gac A as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.

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“…Data were integrated using iMosflm 7.1.146 and scaled with AIMLESS 47. Phases were initially obtained by molecular replacement using Phaser,48 with the structure of PI4KIIIβ bound to PIK‐93 and Rab11 (pdb code: 4D0L) used as the search model.…”

Section: Methodsmentioning

confidence: 99%

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

et al. 2016

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The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.

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iMOSFLM: a new graphical interface for diffraction-image processing withMOSFLM

Cited by 2,780 publications

References 14 publications

Structure of the full-length glucagon class B G-protein-coupled receptor

Structure of the full-length glucagon class B G-protein-coupled receptor

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

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