<i>XDSAPP</i>: a graphical user interface for the convenient processing of diffraction data using<i>XDS</i>

Krug, Michael; Weiss, M.S.; Heinemann, Udo; Muêller, U.

doi:10.1107/s0021889812011715

Cited by 282 publications

(260 citation statements)

References 20 publications

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“…Data were collected at beamline BL14.3 of BESSYII (Berlin, Germany), using a MarMOSAIC 225 CCD detector. The data were processed using XDS (20) as implemented in XDSAPP (21). Data collection and processing statistics are listed in Table 1.…”

Section: Crystallographymentioning

confidence: 99%

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an openunliganded to closed-liganded state during the transport cycle.

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Section: Crystallographymentioning

confidence: 99%

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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“…a part of the physiological substrate histone H3 and human cathepsin L by crystallizing the complex with a catalytic mutant of the enzyme and elucidating its structure [16]. Refinement confirmed the positioning of only three amino acids from the histone H3 [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] peptide. Our crystal packing analysis suggested that there is not enough space to accommodate all 14 peptide residues in the available space in the active site cleft.…”

Section: Abstract: Cathepsin; Cysteine Cathepsin; Substrate Interactionmentioning

confidence: 99%

Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L

Sosnowski

Turk

2016

FEBS Letters

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Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 A revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity.

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“…Crystals appeared within two weeks, were transferred into cryoprotectant (0.1 M MES pH 6.5, 20 mM MnCl 2 , 25 % w/v PEG 8000, 15 % w/v ethylene glycol) and frozen in liquid nitrogen. A 1.95 Å resolution data set was collected at 100 K at BL14.1 operated by the Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring 37 and processed with XDS 38 and POINTLESS. 39 The crystal showed non-merohedral twinning (twinning fraction α = 0.28) and in addition, non-crystallographic translational symmetry (NCS 50 were acquired with different saturation times (0.5, 1, 2 and 4 s).…”

Section: Methodsmentioning

confidence: 99%

Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein–Polysaccharide Interactions

et al. 2016

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Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques because of the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system.

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XDSAPP: a graphical user interface for the convenient processing of diffraction data usingXDS

Cited by 282 publications

References 20 publications

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L

Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein–Polysaccharide Interactions

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