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- EMDB-0153: RELION-3.0 reconstruction of beta-galactosidase particles in EMPI... -

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Basic information

Entry
Database: EMDB / ID: 0153
TitleRELION-3.0 reconstruction of beta-galactosidase particles in EMPIAR-10061
Map dataPostprocessed map. After postprocessing, the map was flipped to get the correct hand.
SampleEscherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG):
SourceEscherichia coli K-12 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 1.89834 Å resolution
AuthorsZivanov J / Nakane T / Scheres SHW
Citation
Journal: Elife / Year: 2018
Title: New tools for automated high-resolution cryo-EM structure determination in RELION-3.
Authors: Jasenko Zivanov / Takanori Nakane / Björn O Forsberg / Dari Kimanius / Wim Jh Hagen / Erik Lindahl / Sjors Hw Scheres
Abstract: Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory ...Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous RELION versions.
#1: Journal: Science / Year: 2015
Title: 2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor.
Authors: Alberto Bartesaghi / Alan Merk / Soojay Banerjee / Doreen Matthies / Xiongwu Wu / Jacqueline L S Milne / Sriram Subramaniam
Abstract: Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli ...Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli β-galactosidase and the cell-permeant inhibitor phenylethyl β-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM.
DateDeposition: Jul 31, 2018 / Header (metadata) release: Oct 24, 2018 / Map release: Dec 26, 2018 / Last update: Dec 26, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.021
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.021
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0153.map.gz (map file in CCP4 format, 364501 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
450 pix
0.64 Å/pix.
= 286.65 Å
450 pix
0.64 Å/pix.
= 286.65 Å
450 pix
0.64 Å/pix.
= 286.65 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.637 Å
Density
Contour Level:0.021 (by author), 0.021 (movie #1):
Minimum - Maximum-0.06469211 - 0.12928325
Average (Standard dev.)-0.0000059097265 (0.004778923)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions450450450
Origin0.00.00.0
Limit449.0449.0449.0
Spacing450450450
CellA=B=C: 286.65002 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6370.6370.637
M x/y/z450450450
origin x/y/z0.0000.0000.000
length x/y/z286.650286.650286.650
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS450450450
D min/max/mean-0.0650.129-0.000

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Supplemental data

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Mask #1

Fileemd_0153_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Escherichia coli beta-galactosidase bound to phenylethyl beta-D-t...

EntireName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Number of components: 2

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Component #1: protein, Escherichia coli beta-galactosidase bound to phenylethyl...

ProteinName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Recombinant expression: No
MassTheoretical: 465 kDa
SourceSpecies: Escherichia coli K-12 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Escherichia coli beta-galactosidase bound to phenylethyl...

ProteinName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: K-12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.3 mg/ml / pH: 8
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 90.15 K / Humidity: 90 % / Details: Blot for 2 seconds before plunging..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 44.84 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 215000.0 X (nominal), 215000.0 X (calibrated)
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 600.0 - 2000.0 nm
Specimen HolderModel: OTHER / Temperature: K ( 79.6 - 79.8 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1487
Details: The pixel size is 0.3185 A / super-resolution pixel.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D2 (2*2 fold dihedral) / Number of projections: 134900
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION
CTF correction: Standard CTF correction inside RELION's reconstruction.
Resolution: 1.89834 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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