EMD-9876

Single-particle
3.1 Å
EMD-9876
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Deposition: 31/03/2019
Map released: 11/09/2019
Last modified: 27/03/2024
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EMD-9876
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EMICSS annotations (xml)

Structure of PaaZ, a bifunctional enzyme in complex with NADP+ and CCoA

EMD-9876

Single-particle
3.1 Å
EMD-9876
3D View Gallery
Deposition: 31/03/2019
Map released: 11/09/2019
Last modified: 27/03/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Method: Single-particle
Aggregation State: Particle
Specimen preparation [1]
Concentration: 0.015 mg/mL
Details: The peak fraction from gel filtration was used for grid preparation. NADP+ and CCoA were added 10 fold excess and incubated for 15 minutes before applying to the grid.
Buffer
pH: 7.4
Buffer components [2]:
Name Formula Concentration ChEBI
HEPES - 25.0 mM -
NACL - 50.0 mM -
Details: Protein was purified and kept in 25mM Hepes buffer and 50 mM NaCl
Grid
Mesh: 300
Model: Quantifoil, UltrAuFoil, R1.2/1.3
Material: GOLD
Details: Grids were glow discharged for 5 minutes and graphene oxide was applied. This was followed by 3 times wash with water and dried.
Pretreatment
Type: GLOW DISCHARGE
Time: {'units': 's', 'valueOf_': '300'}
Atmosphere: AIR
Pressure: 0.02 kPa
Support Film [1]
Material Topology Thickness
GRAPHENE OXIDE CONTINUOUS -
Vitrification
Cryogen name: ETHANE
Chamber humidity: 100%
Chamber temperature: 283.15 K
Instrument: FEI VITROBOT MARK IV
Details: Blotting force 10, blotting time 4 sec, waiting time 15 sec, drying time 0, blotting time 1..
Microscopy [1]
Microscope: FEI TITAN KRIOS
Illumination mode: FLOOD BEAM
Imaging mode: BRIGHT FIELD
Electron source: FIELD EMISSION GUN
Acceleration voltage: 300 kV
C2 aperture diameter: 50.0 µm
Nominal CS: 2.7 mm
Nominal defocus: 2.2 µm - 3.2 µm
Nominal magnification: 75000.0
Calibrated magnification: 134615.0
Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Cooling holder cryogen: NITROGEN
Alignment procedure: COMA FREE
Details: Data was collected with EPU software
Temperature
Minimum: 80.0 K
Maximum: 80.0 K
Image Recording [1]
Detector model: FEI FALCON III (4k x 4k)
Detector mode: COUNTING
Dimensions: 4096 pixel x 4096 pixel
Number of grids: 1
Number of real images: 563
Average exposure time: 60.0 s
Average electron dose per image: 27.0 e/Å2
Details: The 60 second exposure was saved into 75 frames with each frame ~0.36 e-. The frames were then grouped into 3 for alignment and summed images were used for data processing
Image processing [1]
Details: Counting mode
Final reconstruction
Resolution: 3.1 Å ( BY AUTHOR)
Resolution method: FSC 0.143 CUT-OFF
Number of classed used: 1
Number of images used: 120968
Algorithm: BACK PROJECTION
Details:
⌯ Applied Symmetry
Point group: C3
Software [1]
Name Version Details
RELION 2.0 -
Startup model [1]
Type: OTHER
Details:The native map of PaaZ low pass filtered to 60A was used as reference map.
⦨ Initial angle assignment
Type: MAXIMUM LIKELIHOOD
Software [1]
Name Version Details
RELION 2.0 -
⦩ Final angle assignment
Type: MAXIMUM LIKELIHOOD
Software [1]
Name Version Details
RELION 2.0 -
Particle selection [1]
Selected Ref. model Method Software Details
122910 - - - -
Final 3D classification
Software [1]
Name Version Details
RELION 2.0 -
Map
Format: CCP4
Data type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation details: The map of PaaZ is in complex with NADP and Crotonyl CoA
⬡ Geometry
X Y Z
Dimensions 412 412 412
Origin 0 0 0
Spacing 412 412 412
Voxel size 1.04 Å 1.04 Å 1.04 Å
Contour list
Primary Level Source
True 0.085 AUTHOR