EMD-2927

Single-particle
11.5 Å
EMD-2927
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Deposition: 10/03/2015
Map released: 05/08/2015
Last modified: 05/08/2015
Overview 3D View Sample Experiment Validation Additional data Links
Overview 3D View Sample Experiment Validation Additional data Links

EMD-2927
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EMICSS annotations (xml)

Negative stain structure of a type 6 secretion system membrane core complex

EMD-2927

Single-particle
11.5 Å
EMD-2927
3D View Gallery
Deposition: 10/03/2015
Map released: 05/08/2015
Last modified: 05/08/2015
Overview 3D View Sample Experiment Validation Additional data Links
Method: Single-particle
Aggregation State: Particle
Specimen preparation [1]
Concentration: 0.01 mg/mL
Buffer
pH: 8.0
Details: 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.025% w/v Decyl Maltose Neopentyl Glycol (DM-NPG)
Staining
Type: NEGATIVE
Details: Nine microlitres of TssJLM complex sample was applied to glow-discharged carbon-coated copper grids (Agar Scientific). After 30 sec of absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate.
Grid
Details: 400 mesh carbon-coated copper grids
Vitrification
Cryogen name: NONE
Instrument: OTHER
Microscopy [1]
Microscope: FEI TECNAI F20
Illumination mode: FLOOD BEAM
Imaging mode: BRIGHT FIELD
Electron source: FIELD EMISSION GUN
Acceleration voltage: 200 kV
Nominal CS: 2.1 mm
Nominal defocus: -0.5 µm - -2.5 µm
Nominal magnification: 50000.0
Calibrated magnification: 68100.0
Specimen holder model: OTHER
Alignment procedure: LEGACY (Astigmatism: Astigmatism was checked during imaging and during CTF correction of the micrographs. Micrographs is astigmatism >10% were discarded., Electron beam tilt params: )
Image Recording [1]
Detector category: CCD
Detector model: FEI FALCON II (4k x 4k)
Number of real images: 850
Average electron dose per image: 20 e/Å2
Bits per pixel: 16.0
Image processing [1]
Details: A total of 72,146 particles were automatically selected from 1,200 independent images and extracted within boxes of 280 pixels x 280 pixels using EMAN2/BOXER. The CTF was estimated and corrected by phase flipping using EMAN2 (e2ctf). All two- and three-dimensional (2D and 3D) classifications and refinements were performed using RELION 1.3 . We used three rounds of reference-free 2D class averaging to clean up the automatically selected dataset. Only highly populated classes displaying high-resolution features were conserved during this procedure and a final dataset of 26,544 particles was assembled. An initial 3D-model was generated in EMAN2 using using 30 classes. 3D classification was then performed in Relion with 5 classes. The particles corresponding to most populated class (~ 16,738) were used for refinement. Relion auto-refine procedure was used to obtain a final reconstruction at 11.56-A resolution after masking and with C5 symmetry imposed. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion, and FSC curve were corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution .
Final reconstruction
Resolution: 11.5 Å ( BY AUTHOR)
Resolution method: OTHER
Number of images used: 16738
Algorithm: OTHER
Details:
⌯ Applied Symmetry
Point group: C5
Software [1]
Name Version Details
EMAN2, Relion - -
CTF correction
Details: Each particle
Map
Format: CCP4
Data type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation details: Reconstruction of the whole TssJ-TssM-TssL complex
Details: ::::EMDATABANK.org::::EMD-2927::::
⬡ Geometry
X Y Z
Origin 0 0 0
Dimensions (px) 280 280 280
Dimensions (Å) 532.0 532.0 532.0
Voxel size (Å) 1.9 1.9 1.9
Contour list
Primary Level Source
True 0.025 AUTHOR