Real-space refinement in<i>PHENIX</i>for cryo-EM and crystallography

Afonine, Pavel V.; Poon, Billy K.; Read, Randy J.; Sobolev, Oleg; Terwilliger, Thomas C.; Urzhumtsev, Alexandre; Adams, Paul D.

doi:10.1107/s2059798318006551

Cited by 2,263 publications

(1,536 citation statements)

References 85 publications

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“…This resulted in an atomic model of the HCoV-229E ectodomain that included most of the residues between residue 38 and residue 1033, 24 residues before the start of the HR2 region. The atomic model was refined against a half-map using Phenix.real_space_refine (Afonine et al, 2018a) and Rosetta . To improve the geometry of the atomic model, molecular dynamics-assisted model building was carried out in ChimeraX (Goddard et al, 2018) using the ISOLDE tool (Croll, 2018).…”

Section: S-protein Ecto Domain Cryo-em Model Buildingmentioning

confidence: 99%

The human coronavirus HCoV-229E S-protein structure and receptor binding

Tomlinson

Wong

et al. 2019

eLife

173

193

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The coronavirus S-protein mediates receptor binding and fusion of the viral and host cell membranes. In HCoV-229E, its receptor binding domain (RBD) shows extensive sequence variation but how S-protein function is maintained is not understood. Reported are the X-ray crystal structures of Class III-V RBDs in complex with human aminopeptidase N (hAPN), as well as the electron cryomicroscopy structure of the 229E S-protein. The structures show that common core interactions define the specificity for hAPN and that the peripheral RBD sequence variation is accommodated by loop plasticity. The results provide insight into immune evasion and the crossspecies transmission of 229E and related coronaviruses. We also find that the 229E S-protein can expose a portion of its helical core to solvent. This is undoubtedly facilitated by hydrophilic subunit interfaces that we show are conserved among coronaviruses. These interfaces likely play a role in the S-protein conformational changes associated with membrane fusion.Li et al. eLife 2019;8:e51230.

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Section: S-protein Ecto Domain Cryo-em Model Buildingmentioning

confidence: 99%

The human coronavirus HCoV-229E S-protein structure and receptor binding

Tomlinson

Wong

et al. 2019

eLife

173

193

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“…Atomic models for MTP (gp53), Dit (gp58), RBP (gp61), and parts of Tal (gp59), FibL (gp62), FibU (gp68) and TMP (gp57) were built into either the C6 or C3 baseplate reconstructions, using I-TASSER models as starting points [29], complemented with real-space refinement in PHENIX [30]. The RBP, FibL and FibU models were further improved by refinement into focused reconstructions (see Methods).…”

Section: Identification and Modeling Of Tail And Baseplate Proteinsmentioning

confidence: 99%

“…The positions of large aromatic and basic side chains, as well as predicted secondary structures, were compared to the baseplate protein sequences and initial models to confirm the identity of proteins in the baseplate density and to correctly place the models during manual adjustment. The models were iteratively refined using real-space refinement in PHENIX [30], followed by manual correction and local realspace refinement in Coot guided by geometry reports from MolProbity [45] and EMRinger [46]. All models except Tal and TMP were initially refined into the C6 reconstruction.…”

Section: Model Building and Refinementmentioning

confidence: 99%

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

et al. 2020

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Staphylococcus aureus is a common cause of infections in humans. The emergence of virulent, antibiotic-resistant strains of S. aureus is a significant public health concern. Most virulence and resistance factors in S. aureus are encoded by mobile genetic elements, and transduction by bacteriophages represents the main mechanism for horizontal gene transfer. The baseplate is a specialized structure at the tip of bacteriophage tails that plays key roles in host recognition, cell wall penetration, and DNA ejection. We have used high-resolution cryo-electron microscopy to determine the structure of the S. aureus bacteriophage 80α baseplate at 3.75 Å resolution, allowing atomic models to be built for most of the major tail and baseplate proteins, including two tail fibers, the receptor binding protein, and part of the tape measure protein. Our structure provides a structural basis for understanding host recognition, cell wall penetration and DNA ejection in viruses infecting Gram-positive bacteria. Comparison to other phages demonstrates the modular design of baseplate proteins, and the adaptations to the host that take place during the evolution of staphylococci and other pathogens. Author summaryThe emergence of virulent strains of Staphylococcus aureus that are resistant to most antibiotics has become a major public health concern. Virulence and resistance determinants in S. aureus are usually carried on mobile genetic elements (MGEs). Transduction by bacteriophages provides the main means by which MGEs are disseminated horizontally through the bacterial population, and is therefore essential to the evolution of pathogenicity of S. aureus and other pathogens. The baseplate is a complex structure at the tip of bacteriophage tails that serves multiple roles, including host recognition and binding, cell wall penetration, and ejection of the phage DNA. We have determined the structure of the baseplate from bacteriophage 80α, a representative of phages involved in host pathogenicity and in the mobilization of MGEs in S. aureus. Our structure provides a basis for understanding host recognition and infection by phages infecting Gram-positive hosts, and the PLOS Pathogens | https://doi.

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“…The model was refined in real space using Phenix (ref. 59) and the summed map with the refinement resolution limit set to 3.1 Å. Different weights were tested using half maps to check whether the used Phenix refinement protocol shows overfitting to the map ( Supplementary Fig.…”

Section: Atomic Model Buildingmentioning

confidence: 99%

“…Here we describe three cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae that 58 we have determined at near-atomic resolution-an apo and presequence-bound dimeric complex and an 59 apo tetrameric complex. These new structures provide for the first time molecular details of the overall 60 architecture of the TOM complex and, most importantly, the structure of the Tom40 pore and how it 61 engages with presequences.…”

Section: Introduction 22mentioning

confidence: 99%

Cryo-EM structure of the mitochondrial protein-import channel TOM complex from Saccharomyces cerevisiae

Tucker¹,

Park

2019

Preprint

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9Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria 10 following synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria 11 by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. Using cryo-EM, 12we have obtained high-resolution structures of both apo and presequence-bound core TOM complexes 13from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of 14 five proteins arranged in two-fold symmetry-Tom40, a pore-forming β-barrel with an overall negatively-15 charged inner surface, and four auxiliary α-helical transmembrane proteins. The structure suggests that 16 presequences for mitochondrial targeting insert into the Tom40 channel mainly by electrostatic and polar 17interactions. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of 18forming higher-order oligomers. Our study reveals the molecular organization of the TOM complex and 19provides new insights about the mechanism of protein translocation into mitochondria. 20 21

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Real-space refinement inPHENIXfor cryo-EM and crystallography

Abstract: A description is provided of the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite and its application to the re-refinement of cryo-EM-derived models.

Cited by 2,263 publications

References 85 publications

The human coronavirus HCoV-229E S-protein structure and receptor binding

The human coronavirus HCoV-229E S-protein structure and receptor binding

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

Cryo-EM structure of the mitochondrial protein-import channel TOM complex from Saccharomyces cerevisiae

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