X-ray diffraction data were collected on an in-house Rigaku X-ray system with a Saturn 944 CCD detector. Diffraction intensities were indexed, integrated and scaled using the program HKL2000.54 All structure solutions were generated using molecular replacement with PHASER.55 (link) The B chain model of viral substrate product 4A-4B (PDB ID: 3M5M)46 (link) was used as the starting model for all structure solutions. Initial refinement was carried out in the absence of modeled ligand, which was subsequently built in during later stages of refinement. Subsequent crystallographic refinement was carried out within the Phenix program suite, with iterative rounds of TLS or restrained refinement until convergence was achieved.56 (link) 1a3a–VAN complex was refined using the CCP4 program suite.57 (link) The final structures were evaluated with MolProbity58 (link) prior to deposition in the Protein Data Bank. To limit the possibility of model bias throughout the refinement process, 5% of the data were reserved for the free R-value calculation.59 (link) Interactive model building and electron density viewing were carried out using the program COOT.60 (link)
Saturn 944 ccd detector
Manufactured by Rigaku
Sourced in Italy
The Saturn 944 CCD detector is a high-performance X-ray detector developed by Rigaku. It features a large active area and high-resolution imaging capabilities, making it suitable for a wide range of X-ray diffraction and scattering applications.
Automatically generated - may contain errors
Lab products found in correlation
Fr e superbright generator, by Rigaku (3 mentions)
Micromax 007hf generator, by Rigaku (2 mentions)
X ray system, by Rigaku (1 mentions)
Micromax 007hf cu, by Rigaku (1 mentions)
Hkl2000, by Rigaku (1 mentions)
Micromax 007hf, by Rigaku (1 mentions)
Sparse matrix screens, by Molecular Dimensions (1 mentions)
Mosquito liquid handler, by SPT Labtech (1 mentions)
Mx300 ccd detector, by Rayonix (1 mentions)
Rnase a, by Merck Group (1 mentions)
11 protocols using saturn 944 ccd detector
1
X-ray Crystallographic Structure Determination
2
Crystallization of PaFabA-N42FTA Complex
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N42FTA (1 mM) was incubated for 1 h with PaFabA at 7 mg/ml before setting the plates. Crystals appeared at 20 °C after 2 days from a hanging drop of 2 μl of protein solution with 1 μl of reservoir solution containing 0.1 M lithium sulfate, 12% polyethylene glycol MME 5000, and 0.1 M sodium citrate (pH 5). Crystals were cryoprotected by supplementing the mother liquor with 15% glycerol.
Data were collected in-house using a Rigaku Micromax™-007HF Cu anode with VariMax optics and a Rigaku Saturn 944 + CCD detector. Data were processed with xia2 [46] , [47] , [48] (link), [49] , [50] (link). The structure was solved using the coordinates of the wild-type PaFabA [42] (link) and the programme Phaser [51] (link). PHENIX [52] (link) was used for autobuilding from molecular replacement solution. The model was adjusted with Coot [53] (link) after each round of refinement in REFMAC 5.6 from the CCP4 programme suite 6.2.0 with local NCS restraints (option in programme) and TLS parameters [54] (link) applied throughout. N42FTA was added to the model when the difference electron density was clear (Fig. 6 a). Coordinates and topologies of ligand were generated by PRODRG [55] (link). The quality of the structure was checked with MolProbity [56] (link). Final refinement statistics are given in Table 1 . Figures were drawn using PyMOL [57] .
Data were collected in-house using a Rigaku Micromax™-007HF Cu anode with VariMax optics and a Rigaku Saturn 944 + CCD detector. Data were processed with xia2 [46] , [47] , [48] (link), [49] , [50] (link). The structure was solved using the coordinates of the wild-type PaFabA [42] (link) and the programme Phaser [51] (link). PHENIX [52] (link) was used for autobuilding from molecular replacement solution. The model was adjusted with Coot [53] (link) after each round of refinement in REFMAC 5.6 from the CCP4 programme suite 6.2.0 with local NCS restraints (option in programme) and TLS parameters [54] (link) applied throughout. N42FTA was added to the model when the difference electron density was clear (
3
Structural Determination of C. thermophilum Vps29
4
Structural Analysis of GAR Protein
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GAR crystals were tested and screened on an in-house X-ray source of Rigaku MicroMax-002+ system equipped with Saturn 944+ CCD detector (Rigaku) and Oxford Cryo-system. The data sets were integrated and processed using Rigaku crystalclear-1.44 for in-house data sets and HKL2000 for Synchrotron. High-resolution native data sets were collected at 100 K on beamline BL17U of Shanghai Synchrotron Radiation Facility, the crystal structure of GAR was determined by single isomorphous replacement with anomalous scattering of mercury derivative and refined to 1.5 Å.
Heavy atoms searching and initial Phasing were undertaken by SHELXC/D/E and atomic model was auto-built PHENIX suite, model improvement and refinements were finalized in COOT and REFMAC in CCP4. In the final model, the electron density is clear and continuous, 98.5% residues located in favoured region, 2.5% located in allowed region and no residues located in outlier region by Ramachandran plot. All crystallographic figures were drawn in Pymol.
Heavy atoms searching and initial Phasing were undertaken by SHELXC/D/E and atomic model was auto-built PHENIX suite, model improvement and refinements were finalized in COOT and REFMAC in CCP4. In the final model, the electron density is clear and continuous, 98.5% residues located in favoured region, 2.5% located in allowed region and no residues located in outlier region by Ramachandran plot. All crystallographic figures were drawn in Pymol.
5
Crystallization of Aspartate Synthase from Mycobacterium smegmatis
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Prior to setting up crystallisation experiments, protein was dialysed into 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 10% glycerol, 100 µM EDTA and 1 mM DTT and concentrated by ultrafiltration. Crystals of Ms-AspS were obtained by vapour diffusion in 96-well sitting drop plates (SwissSci), using a Mosquito liquid handler (TTP Labtech) to pipette droplets of 100 nl Ms-AspS (25 mg/ml) +100 nl reservoir solution. Initial crystals were obtained from commercial sparse matrix screens (Molecular Dimensions) grown over a reservoir of 30% (v/v) pentaerythritol ethoxylate, 0.1 M magnesium formate, 0.1 M Tris-HCl, pH 8.5. Crystals were briefly immersed in reservoir solution complemented with 15% glycerol for cryoprotection, mounted in nylon loops and flash frozen in liquid nitrogen. X-ray diffraction data was recorded on a MicroMax 007HF X-ray generator equipped with a Saturn 944 CCD detector (Rigaku), integrated and scaled using XDS, XSCALE [46] (link). Initial phases were obtained by molecular replacement (PHASER, search model pdb entry 1C0A) [47] (link). The model was rebuilt and refined [48] (link), [49] (link). Coordinates and structure factors for Ms-AspS are deposited in the Protein Data Bank (www.rcsb.org ).
6
Cryo-protected Carbanpenem Drug Soaked Crystals
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LdtMt2-apo, co-crystals as well as crystals soaked with carbapenem drugs were cryo-protected in 30% glycerol, 20% 5000 MME and 120 mM ammonium sulfate buffer and flash cooled in liquid nitrogen. For LdtMt2-apo and co-crystals with faropenem and doripenem, X-ray diffraction data were collected at a wavelength of 1.54 Å using an in-house CuKα X-ray source (Rigaku FR-E+ SuperBright generator with a Saturn 944+ CCD Detector; Rigaku). for LdtMt2 apo and crystals soaked with evolved the carbapenems T206, T208 and T210, diffraction data were collected at 100K at a wavelength of 0.98 Å on beamline 19-ID at the Advanced Photon Source (Argonne National Laboratory) and the diffraction data were recorded on an ADSC Quantum 315r CCD detector, and processed with HKL3000 38 (link).
LdtMt1-apo and co-crystal with faropenem were cryoprotected with 30% PEG8000, 10% PEG6000 and 100 mM Bicine pH 9.0 buffer and flash cooled in liquid nitrogen. The X-ray diffraction data were collected and analyzed as described below.
LdtMt1-apo and co-crystal with faropenem were cryoprotected with 30% PEG8000, 10% PEG6000 and 100 mM Bicine pH 9.0 buffer and flash cooled in liquid nitrogen. The X-ray diffraction data were collected and analyzed as described below.
7
Peptide 19B Crystal Structure Determination
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Peptide 19B was dissolved in 50% CH3OH with 10 mg/mL and crystallized at 25 °C using sitting drop vapor diffusion method against reservoir solution of 90% CH3OH. Crystals appeared, attained full size within 20 days, and then started to decay after four weeks. Crystals were flash-frozen in liquid nitrogen and cryo-protected by 30% Glycerol in mother liquor. The crystals were screened and collected at 100 K by in-house X-ray diffraction system equipped with high-intensity sealed Copper tube X-ray generator (Rigaku® MicroMax-002+), an AFC11 goniometer, a Saturn 944+ CCD detector (Rigaku®) and an Oxford Cryo-system.
8
Cryo-protected Carbanpenem Drug Soaked Crystals
9
Crystallization and Structure Determination of sDrl-2
10
Protein Structure Determination by Diffraction
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For structure determination, diffraction data were collected from a single crystal at 100 K using synchrotron radiation (Canadian Light Source, Saskatoon, SK, Canada) and a Rayonix MX300 CCD detector. For exploiting the anomalous signal from P atoms, additional data were collected at home from another single crystal using a MicroMax-007 HF generator (Rigaku) and a Saturn 944+ CCD detector (Rigaku). Both data sets were processed with the xia2 package (Winter et al., 2013 ▶ ); for the home-source data the Friedel mates were not merged together, unlike for the synchrotron data. The structure model was initially built by a difference Fourier method with a ligand/solvent-omitted starting model generated from PDB entry 4h5d (Park et al., 2012 ▶ ). The model was improved through iterative rounds of manual and automated refinement with Coot (Emsley et al., 2010 ▶ ) and REFMAC5 (Murshudov et al., 2011 ▶ ). The final model was deposited in the Protein Data Bank (PDB entry 4lfv ). Data-collection and refinement statistics are presented in Table 1 ▶ .
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