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- EMDB-39808: E.coli. beta-galactosidase -

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Basic information

Entry
Database: EMDB / ID: EMD-39808
TitleE.coli. beta-galactosidase
Map datarelion sharpened map
Sample
  • Complex: E. coli. beta-galactosidase
Keywordshydrolase / enzyme
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYadav S / Vinothkumar KR
Funding support India, 2 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Other governmentRTI4006
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2024
Title: Factors affecting macromolecule orientations in thin films formed in cryo-EM.
Authors: Swati Yadav / Kutti R Vinothkumar /
Abstract: The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the ...The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.
History
DepositionApr 19, 2024-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBj / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_39808.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrelion sharpened map
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.23099032 - 0.39067167
Average (Standard dev.)-0.000009177171 (±0.011932213)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 342.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : E. coli. beta-galactosidase

EntireName: E. coli. beta-galactosidase
Components
  • Complex: E. coli. beta-galactosidase

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Supramolecule #1: E. coli. beta-galactosidase

SupramoleculeName: E. coli. beta-galactosidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 466 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMC4H11NO3Tris
2.5 mMMgCl2magnesium chloride
5.0 mMCaCl2calcium chloride
2.0 mMC2H6OS2-Mercaptoethanol
200.0 mMNaClsodium chloride
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Average exposure time: 60.0 sec. / Average electron dose: 24.0 e/Å2
Details: Images were collected in movie mode @40 frames per second. Total of 24 frames were saved.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130841 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Initial model is generated with SGD in Relion
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 200000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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