Journal: Ultramicroscopy / Year: 2014 Title: CTER-rapid estimation of CTF parameters with error assessment. Authors: Pawel A Penczek / Jia Fang / Xueming Li / Yifan Cheng / Justus Loerke / Christian M T Spahn / Abstract: In structural electron microscopy, the accurate estimation of the Contrast Transfer Function (CTF) parameters, particularly defocus and astigmatism, is of utmost importance for both initial ...In structural electron microscopy, the accurate estimation of the Contrast Transfer Function (CTF) parameters, particularly defocus and astigmatism, is of utmost importance for both initial evaluation of micrograph quality and for subsequent structure determination. Due to increases in the rate of data collection on modern microscopes equipped with new generation cameras, it is also important that the CTF estimation can be done rapidly and with minimal user intervention. Finally, in order to minimize the necessity for manual screening of the micrographs by a user it is necessary to provide an assessment of the errors of fitted parameters values. In this work we introduce CTER, a CTF parameters estimation method distinguished by its computational efficiency. The efficiency of the method makes it suitable for high-throughput EM data collection, and enables the use of a statistical resampling technique, bootstrap, that yields standard deviations of estimated defocus and astigmatism amplitude and angle, thus facilitating the automation of the process of screening out inferior micrograph data. Furthermore, CTER also outputs the spatial frequency limit imposed by reciprocal space aliasing of the discrete form of the CTF and the finite window size. We demonstrate the efficiency and accuracy of CTER using a data set collected on a 300kV Tecnai Polara (FEI) using the K2 Summit DED camera in super-resolution counting mode. Using CTER we obtained a structure of the 80S ribosome whose large subunit had a resolution of 4.03Å without, and 3.85Å with, inclusion of astigmatism parameters.
History
Deposition
Jan 22, 2014
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Header (metadata) release
Mar 19, 2014
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Map release
Jan 21, 2015
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Update
Jan 21, 2015
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Current status
Jan 21, 2015
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_5889.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
human 80S ribosome
Voxel size
X=Y=Z: 1.264 Å
Density
Contour Level
By AUTHOR: 8.75 / Movie #1: 8.75
Minimum - Maximum
-6.2626152 - 48.298103330000004
Average (Standard dev.)
0.60599691 (±2.57134986)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
280
280
280
Spacing
280
280
280
Cell
A=B=C: 353.92 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.264
1.264
1.264
M x/y/z
280
280
280
origin x/y/z
0.000
0.000
0.000
length x/y/z
353.920
353.920
353.920
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-95
-75
153
NX/NY/NZ
200
200
200
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
280
280
280
D min/max/mean
-6.263
48.298
0.606
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Supplemental data
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Sample components
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Entire : human 80S ribosome
Entire
Name: human 80S ribosome
Components
Sample: human 80S ribosome
Complex: 80S ribosome
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Supramolecule #1000: human 80S ribosome
Supramolecule
Name: human 80S ribosome / type: sample / ID: 1000 / Details: sample was monodisperse / Oligomeric state: 1 / Number unique components: 1
Molecular weight
Theoretical: 4.2 MDa
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Supramolecule #1: 80S ribosome
Supramolecule
Name: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)
Organism: Homo sapiens (human) / synonym: human / Tissue: kidney / Cell: HEK293 / Organelle: cytosol
Molecular weight
Theoretical: 4.2 MDa
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.5 Details: 20 mM HEPES, pH 7.5, 100 mM KCl, 1.5 mM MgCl2, 0.5 mM spermidine, 0.04 mM spermine, 1 mM DTT
Grid
Details: Quantifoil 2/4 Cu/Re with custom continuous carbon film
Vitrification
Cryogen name: ETHANE / Instrument: OTHER
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Electron microscopy
Microscope
FEI TECNAI F30
Date
Feb 12, 2013
Image recording
Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 893 Details: The data was collected in movie mode at 39,000x magnification. Frames 3 to 20 were decimated twice, motion corrected with UCSHImage4, and averaged.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm
Sample stage
Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
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Image processing
CTF correction
Details: Per-particle
Final reconstruction
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: OTHER / Software - Name: SPARX Details: The resolution reported is for the large subunit (60S) only. Number images used: 35198
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