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- EMDB-5995: Structure of beta-galactosidase at 3.2-A resolution obtained by c... -

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Basic information

Entry
Database: EMDB / ID: EMD-5995
TitleStructure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
Map dataReconstruction of beta-galactosidase
Sample
  • Sample: Escherichia coli beta-galactosidase
  • Protein or peptide: beta-galactosidase
Keywordsatomic resolution cryo-electron microscopy / single-particle EM / direct electron detectors / 3D reconstruction / frame alignment / CTF determination / structure refinement / radiation damage / protein complexes / enzyme active site structure
Function / homology
Function and homology information


alkali metal ion binding / lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding / magnesium ion binding / identical protein binding
Similarity search - Function
Glycoside hydrolase, family 2, beta-galactosidase / Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site ...Glycoside hydrolase, family 2, beta-galactosidase / Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site / Glycosyl hydrolases family 2 signature 1. / Glycoside hydrolase, family 2 / Glycosyl hydrolases family 2, sugar binding domain / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Glycoside hydrolase-type carbohydrate-binding / Galactose mutarotase-like domain superfamily / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBartesaghi A / Matthies D / Banerjee S / Merk A / Subramaniam S
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy.
Authors: Alberto Bartesaghi / Doreen Matthies / Soojay Banerjee / Alan Merk / Sriram Subramaniam /
Abstract: We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side ...We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg(2+) ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e(-)/Å(2). In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.
History
DepositionJun 24, 2014-
Header (metadata) releaseJul 30, 2014-
Map releaseJul 30, 2014-
UpdateOct 7, 2015-
Current statusOct 7, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0224
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0224
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j7h
  • Surface level: 0.0224
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3j7h
  • Surface level: 0.0224
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5995.gif

Downloads & links

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Map

FileDownload / File: emd_5995.map.gz / Format: CCP4 / Size: 146.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of beta-galactosidase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.64 Å/pix.
x 340 pix.
= 216.75 Å		0.64 Å/pix.
x 340 pix.
= 216.75 Å		0.64 Å/pix.
x 340 pix.
= 216.75 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.6375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0224 / Movie #1: 0.0224
Minimum - Maximum-0.05132602 - 0.08781078
Average (Standard dev.)0.00028028 (±0.00593622)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 216.75 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.63750.63750.6375
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z216.750216.750216.750
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean-0.0510.0880.000

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Supplemental data

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Supplemental map: emd 5995 additional 1.map

Fileemd_5995_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: emd 5995 half map 1.map

Fileemd_5995_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: emd 5995 half map 2.map

Fileemd_5995_half_map_2.map
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

EntireName: Escherichia coli beta-galactosidase
Components
  • Sample: Escherichia coli beta-galactosidase
  • Protein or peptide: beta-galactosidase

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Supramolecule #1000: Escherichia coli beta-galactosidase

SupramoleculeName: Escherichia coli beta-galactosidase / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: tetramer / Number unique components: 1
Molecular weightTheoretical: 465 KDa
Method: ProtParam tool: Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A., Protein Identification and Analysis Tools on the ExPASy Server, (in) John M. Walker ...Method: ProtParam tool: Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A., Protein Identification and Analysis Tools on the ExPASy Server, (in) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607

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Macromolecule #1: beta-galactosidase

MacromoleculeName: beta-galactosidase / type: protein_or_peptide / ID: 1 / Name.synonym: beta-gal, b-gal / Number of copies: 1 / Oligomeric state: tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: cytoplasm
Molecular weightTheoretical: 465 KDa
SequenceUniProtKB: Beta-galactosidase / GO: beta-galactosidase activity

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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
Details: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP
GridDetails: 200 mesh Quantifoil R2/2 grids, plasma cleaned
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90.15 K / Instrument: LEICA EM GP / Method: Blot for 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 79.6 K / Max: 79.8 K / Average: 79.7 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 105,000 times magnification.
Legacy - Electron beam tilt params: 5
Specialist opticsEnergy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DetailsParallel beam illumination
DateOct 31, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 509 / Average electron dose: 45 e/Å2
Details: Every image is the average of 38 frames recorded by the direct electron detector. The complete set of electron micrographs used to obtain the density map presented here is available through ...Details: Every image is the average of 38 frames recorded by the direct electron detector. The complete set of electron micrographs used to obtain the density map presented here is available through the Electron Microscopy Pilot Image ARchive (EMPIAR).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsIndividual frames of each movie were aligned by cross-correlation using the cumulative average of previously aligned frames as a reference to align the remaining frames. Parameters of the contrast transfer function for each micrograph were estimated from power spectra obtained using periodogram averaging with tiles of size 512x512 pixels extracted from all frames of each movie. These power spectra were then radially averaged and used to estimate the defocus for each image using frequencies in the 15.0-3.0 Angstrom range. 24,750 particles were picked automatically from the best 509 micrographs by detecting the local maxima of correlation of each image with a Gaussian disk of 100 Angstrom in radius and extracted using a binning factor of 2 and a box size of 384x384 pixels. Particles were then subjected to reference-free 2D classification in EMAN2. 160 classes out of a total of 250 were used for de novo initial model determination using e2initialmodel.py and imposing D2 symmetry. A subset of 23,452 particles (corresponding to the 160 classes used for the initial model building) was then used for 3D refinement using a gold-standard approach. Two stacks of 11,726 particles each were independently subjected to eight rounds of iterative refinement in FREALIGN using a high-resolution frequency limit of 8 Angstrom and using the best 50% of particles from each stack according to phase residual values (equivalent to 5,863 particles) to calculate the reconstructions at each iteration. At this point particles were re-extracted from the original unbinned micrographs using a box size of 768x768 pixels and further refined in FREALIGN starting from the most recent set of alignments obtained with the binned data. The final map was obtained by averaging the two half-reconstructions in real space and corrected by a B-factor of -85 Angstrom^2 for the purpose of visualization.
CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: OTHER / Software - Name: FREALIGN, EMAN2 / Number images used: 11726
FSC plot (resolution estimation)

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