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- EMDB-5995: Structure of beta-galactosidase at 3.2-A resolution obtained by c... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-5995 | |||||||||
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Title | Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy | |||||||||
![]() | Reconstruction of beta-galactosidase | |||||||||
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![]() | atomic 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 | ![]() 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 | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
![]() | Bartesaghi A / Matthies D / Banerjee S / Merk A / Subramaniam S | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 136.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.4 KB 13.4 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 13.3 KB | Display | ![]() |
Images | ![]() ![]() | 82.5 KB 4.5 KB | ||
Others | ![]() ![]() ![]() | 59.7 MB 61.2 MB 61.2 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3j7hMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 108.0 Data #1: Beta Galactosidase micrographs [micrographs - single frame]) ![]() Data size: 442.5 Data #1: Full set of multi-frame micrographs [micrographs - multiframe] Data #2: Sample set of multi-frame micrographs [micrographs - multiframe]) |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of beta-galactosidase | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.6375 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Supplemental map: emd 5995 additional 1.map
File | emd_5995_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Supplemental map: emd 5995 half map 1.map
File | emd_5995_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Supplemental map: emd 5995 half map 2.map
File | emd_5995_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Escherichia coli beta-galactosidase
Entire | Name: Escherichia coli beta-galactosidase |
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Components |
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-Supramolecule #1000: Escherichia coli beta-galactosidase
Supramolecule | Name: Escherichia coli beta-galactosidase / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: tetramer / Number unique components: 1 |
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Molecular weight | Theoretical: 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 |
-Macromolecule #1: beta-galactosidase
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 465 KDa |
Sequence | UniProtKB: Beta-galactosidase / GO: beta-galactosidase activity |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 2.3 mg/mL |
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Buffer | pH: 8 Details: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP |
Grid | Details: 200 mesh Quantifoil R2/2 grids, plasma cleaned |
Vitrification | Cryogen 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
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 79.6 K / Max: 79.8 K / Average: 79.7 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 105,000 times magnification. Legacy - Electron beam tilt params: 5 |
Specialist optics | Energy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Details | Parallel beam illumination |
Date | Oct 31, 2013 |
Image recording | Category: 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 beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated 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 stage | Specimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |