Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp15076210
Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp20050030
Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp22018027
Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp23120525
Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp25120725
Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp15H01647
Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp17H05891
Japan
Citation
Journal: To Be Published Title: Mechanism for drastic reduction in catalytic activity of Trp89Phe-mutated glutamate dehydrogenase revealed by cryoEM-sampling metastable conformation in action Authors: Wakabayashi T / Oide M / Matsui Y / Nakasako M
Details: 0.5 mM NADP, 100 mM Glutamate in 5 mM Tris-HCl at pH7.5.
Grid
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa Details: Both sides of the grid were glow-discharged for 45 s at 20 mA and 20 Pa.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV Details: The sample solution was flash-frozen 2-h after mixing the protein solution and buffer solution..
Details
8.96 mg/mL GDH W89F
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Electron microscopy
Microscope
JEOL CRYO ARM 300
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7651 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.35000000000000003 µm
Sample stage
Cooling holder cryogen: NITROGEN
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Image processing
Particle selection
Number selected: 3534400
Startup model
Type of model: INSILICO MODEL Details: The startup model was generated by de novo 3D model generation in Relion.
Final reconstruction
Number classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 119274
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classification
Number classes: 32 / Avg.num./class: 115000 / Software - Name: RELION (ver. 4.0) Details: The final 3D classification carried out by two-step process. In the first, all particles were separated into eight classes. Subsequently, each of these classes was further classified into ...Details: The final 3D classification carried out by two-step process. In the first, all particles were separated into eight classes. Subsequently, each of these classes was further classified into four subclasses. This entire process was conducted following a symmetry expansion operation on all particles, in accordance with the D3 symmetry of GDH.
FSC plot (resolution estimation)
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