<i>PHENIX</i>: building new software for automated crystallographic structure determination

Adams, Paul D.; Grosse‐Kunstleve, Ralf W.; Hung, Li Wei; Ioerger, Thomas R.; McCoy, Airlie J.; Moriarty, Nigel W.; Read, Randy J.; Sacchettini, James C.; Sauter, Nicholas K.; Terwilliger, Thomas C.

doi:10.1107/s0907444902016657

Cited by 4,267 publications

(4,167 citation statements)

References 18 publications

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“…Refinement was performed using phenix.refine. 35 The structure was hand-corrected with Coot. 36 Later refinement included TLS refinement (using TLSMD 37 parameters), followed by a final refinement using hydrogens added with Molprobity.…”

Section: Resultsmentioning

confidence: 99%

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

et al. 2010

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Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild-type Rop is a homodimeric four-helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native state, Rop has two buried, reduced cysteine residues in its core, but these are prone to oxidation in destabilized variants, particularly upon extended storage. To circumvent this problem, we designed and characterized a Cys-free variant of Rop, including solving the 2.3 Å X-ray crystal structure. We show that the C38A C52V variant has similar structure, stability and in vivo activity to wild-type Rop, but that it has dramatically faster unfolding kinetics like virtually every other mutant of Rop that has been characterized. This cysteine-free Rop has already proven useful for studies on solution topology and on the relationship of core mutations to stability. It also suggests a general strategy for removal of pairs of Cys residues in proteins, both to make them more experimentally tractable and to improve their storage properties for therapeutic or industrial purposes.

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Section: Resultsmentioning

confidence: 99%

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

et al. 2010

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“…A full‐length model for human RAD51 was built, using PDB entry 1SZP for the RAD51 N‐terminal domain and 1N0W for modelling hRAD51's interdomain linker sequence. The structure was refined in Phenix (Adams et al , 2002) to a resolution of 3.9 Å, using Coot (Emsley et al , 2010) for model building and applying NCS restraints to the ATPase and N‐terminal domains of the 14 hRAD51 molecules in the asymmetric unit.…”

Section: Methodsmentioning

confidence: 99%

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

et al. 2018

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An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

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“…Phaser under Phenix (Adams et al , 2002) was able to locate the TMD and NBD with good packing scores. Similar solution could also be obtained using MOLREP in CCP4.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

et al. 2017

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Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.

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PHENIX: building new software for automated crystallographic structure determination

Cited by 4,267 publications

References 18 publications

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

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