Towards automated crystallographic structure refinement with <i>phenix.refine</i>

Afonine, Pavel V.; Grosse‐Kunstleve, Ralf W.; Echols, Nathaniel; Headd, Jeffrey J.; Moriarty, Nigel W.; Mustyakimov, Marat; Terwilliger, Thomas C.; Urzhumtsev, Alexandre; Zwart, Peter H.; Adams, Paul D.

doi:10.1107/s0907444912001308

Cited by 4,933 publications

(4,549 citation statements)

References 122 publications

Supporting

Mentioning

4,406

Contrasting

Unclassified

Order By: Relevance

“…However, most of these were not well separated from other potential solutions in their respective searches, typically with two to four other candidate solutions giving LLG values at least 75% that of the top solution. The models giving the top 2 solutions (as judged by both TF-Z and LLG metrics) were used as a single ensemble for phase calculation followed by manual building using COOT 38 and reciprocal space refinement using phenix.refine 39 . Manual refinement was complemented by the use of ROSETTA refinement in Phenix 13 .…”

Section: Methodsmentioning

confidence: 99%

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Sjodt

Brock

Dobihal

et al. 2018

Nature

119

139

View full text Add to dashboard Cite

The Shape, Elongation, Division, and Sporulation (“SEDS”) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was long enigmatic, but recent work1–3 has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase – a role previously attributed exclusively to members of the penicillin binding protein family4. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis for SEDS activity, and no structural data are available for RodA or any homolog thereof. Here, we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a novel ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating it is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

show abstract

Section: Methodsmentioning

confidence: 99%

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Sjodt

Brock

Dobihal

et al. 2018

Nature

119

139

View full text Add to dashboard Cite

show abstract

“…To address the issue of potential model bias, molecular replacement was repeated with H2A–H2B omitted from the search model. Each of the 24 protein chains were defined as individual rigid bodies and their position refined by rigid body refinement using the L–BFGS optimization method as implemented in phenix.refine (Afonine et al , 2012). B factors from the high‐resolution search models were maintained during the initial cycles of refinement and the initial model was used as both the starting and reference model for subsequent Deformable Elastic Network (DEN) refinement using CNS over a grid‐enabled web server hosted by SBGrid (Schroder et al , 2010; O'Donovan et al , 2012).…”

Section: Methodsmentioning

confidence: 99%

Structural evidence for Nap1‐dependent H2A–H2B deposition and nucleosome assembly

et al. 2016

View full text Add to dashboard Cite

Nap1 is a histone chaperone involved in the nuclear import of H2A–H2B and nucleosome assembly. Here, we report the crystal structure of Nap1 bound to H2A–H2B together with in vitro and in vivo functional studies that elucidate the principles underlying Nap1‐mediated H2A–H2B chaperoning and nucleosome assembly. A Nap1 dimer provides an acidic binding surface and asymmetrically engages a single H2A–H2B heterodimer. Oligomerization of the Nap1–H2A–H2B complex results in burial of surfaces required for deposition of H2A–H2B into nucleosomes. Chromatin immunoprecipitation‐exonuclease (ChIP‐exo) analysis shows that Nap1 is required for H2A–H2B deposition across the genome. Mutants that interfere with Nap1 oligomerization exhibit severe nucleosome assembly defects showing that oligomerization is essential for the chaperone function. These findings establish the molecular basis for Nap1‐mediated H2A–H2B deposition and nucleosome assembly.

show abstract

“…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. The final model of Apo PI4KIIIβ bound to Rab11 was built using iterative model building in COOT49 and refinement using Phenix50, 51 to R work = 21.6 and R free = 24.6. The final model of PI4KIIIβ bound to BQR695 in complex with GDP loaded Rab11 was refined to R work =25.5 and R free = 28.6.…”

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

View full text Add to dashboard Cite

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.

show abstract

Towards automated crystallographic structure refinement with phenix.refine

Cited by 4,933 publications

References 122 publications

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Structural evidence for Nap1‐dependent H2A–H2B deposition and nucleosome assembly

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

Contact Info

Product

Resources

About