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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: EMD-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.
Sample
  • Complex: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
    • Protein or peptide: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.9 Å
AuthorsZivanov J / Nakane T / Scheres SHW
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_A025_1013 United Kingdom
CitationJournal: 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 β- ...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.
History
DepositionJul 31, 2018-
Header (metadata) releaseOct 24, 2018-
Map releaseDec 26, 2018-
UpdateApr 28, 2021-
Current statusApr 28, 2021Processing site: PDBe / Status: Released

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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

emd_0153.png

Downloads & links

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Map

FileDownload / File: emd_0153.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map. After postprocessing, the map was flipped to get the correct hand.
Voxel sizeX=Y=Z: 0.637 Å
Density
Contour LevelBy AUTHOR: 0.021 / Movie #1: 0.021
Minimum - Maximum-0.06469211 - 0.12928325
Average (Standard dev.)-5.9097265e-06 (±0.004778923)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 286.65002 Å
α=β=γ: 90.0 °

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
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

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 2. Since the refinement was...

Fileemd_0153_half_map_1.map
AnnotationUnfiltered half map 2. Since the refinement was performed in the opposite hand, the half map was flipped before deposition.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 1. Since the refinement was...

Fileemd_0153_half_map_2.map
AnnotationUnfiltered half map 1. Since the refinement was performed in the opposite hand, the half map was flipped before deposition.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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)
Components
  • Complex: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
    • Protein or peptide: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)

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Supramolecule #1: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-t...

SupramoleculeName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: cytoplasm
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 465 KDa

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Macromolecule #1: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-t...

MacromoleculeName: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: beta-galactosidase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MITDSLAVVL QRRDWENPGV TQLNRLAAHP PFASWRNSEE ARTDRPSQQL RSLNGEWRFA WFPAPEAVPE SWLECDLPEA DTVVVPSNW QMHGYDAPIY TNVTYPITVN PPFVPTENPT GCYSLTFNVD ESWLQEGQTR IIFDGVNSAF HLWCNGRWVG Y GQDSRLPS ...String:
MITDSLAVVL QRRDWENPGV TQLNRLAAHP PFASWRNSEE ARTDRPSQQL RSLNGEWRFA WFPAPEAVPE SWLECDLPEA DTVVVPSNW QMHGYDAPIY TNVTYPITVN PPFVPTENPT GCYSLTFNVD ESWLQEGQTR IIFDGVNSAF HLWCNGRWVG Y GQDSRLPS EFDLSAFLRA GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDFHVAT RFNDDFSRAV LE AEVQMCG ELRDYLRVTV SLWQGETQVA SGTAPFGGEI IDERGGYADR VTLRLNVENP KLWSAEIPNL YRAVVELHTA DGT LIEAEA CDVGFREVRI ENGLLLLNGK PLLIRGVNRH EHHPLHGQVM DEQTMVQDIL LMKQNNFNAV RCSHYPNHPL WYTL CDRYG LYVVDEANIE THGMVPMNRL TDDPRWLPAM SERVTRMVQR DRNHPSVIIW SLGNESGHGA NHDALYRWIK SVDPS RPVQ YEGGGADTTA TDIICPMYAR VDEDQPFPAV PKWSIKKWLS LPGETRPLIL CEYAHAMGNS LGGFAKYWQA FRQYPR LQG GFVWDWVDQS LIKYDENGNP WSAYGGDFGD TPNDRQFCMN GLVFADRTPH PALTEAKHQQ QFFQFRLSGQ TIEVTSE YL FRHSDNELLH WMVALDGKPL ASGEVPLDVA PQGKQLIELP ELPQPESAGQ LWLTVRVVQP NATAWSEAGH ISAWQQWR L AENLSVTLPA ASHAIPHLTT SEMDFCIELG NKRWQFNRQS GFLSQMWIGD KKQLLTPLRD QFTRAPLDND IGVSEATRI DPNAWVERWK AAGHYQAEAA LLQCTADTLA DAVLITTAHA WQHQGKTLFI SRKTYRIDGS GQMAITVDVE VASDTPHPAR IGLNCQLAQ VAERVNWLGL GPQENYPDRL TAACFDRWDL PLSDMYTPYV FPSENGLRCG TRELNYGPHQ WRGDFQFNIS R YSQQQLME TSHRHLLHAE EGTWLNIDGF HMGIGGDDSW SPSVSAEFQL SAGRYHYQLV WCQK

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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

Concentration2.3 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90.15 K / Instrument: LEICA EM GP / Details: Blot for 2 seconds before plunging..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 215000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 215000
TemperatureMin: 79.6 K / Max: 79.8 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 1-38 / Number real images: 1487 / Average electron dose: 44.84 e/Å2
Details: The pixel size is 0.3185 A / super-resolution pixel.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 229553
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4.1.1.0)
RELION (ver. 3.0)CTF refinement

Details: Standard CTF correction inside RELION's reconstruction.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: RELION SGD
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 134900
FSC plot (resolution estimation)

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