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Yorodumi- PDB-8bk8: Cryo-EM structure of beta-galactosidase at 2.9 A resolution plung... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8bk8 | ||||||||||||
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Title | Cryo-EM structure of beta-galactosidase at 2.9 A resolution plunged 205 ms after mixing with apoferritin | ||||||||||||
Components | Beta-galactosidase | ||||||||||||
Keywords | HYDROLASE / Glycosyl hydrolase | ||||||||||||
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 | ||||||||||||
Biological species | Escherichia coli (E. coli) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||||||||
Authors | Torino, S. / Dhurandhar, M. / Efremov, R. | ||||||||||||
Funding support | European Union, Belgium, 3items
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Citation | Journal: Nat Methods / Year: 2023 Title: Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting. Authors: Stefania Torino / Mugdha Dhurandhar / Annelore Stroobants / Raf Claessens / Rouslan G Efremov / Abstract: Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient ...Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid reaction initiation and plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8bk8.cif.gz | 703 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8bk8.ent.gz | 575.1 KB | Display | PDB format |
PDBx/mmJSON format | 8bk8.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8bk8_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 8bk8_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8bk8_validation.xml.gz | 107.4 KB | Display | |
Data in CIF | 8bk8_validation.cif.gz | 163 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bk/8bk8 ftp://data.pdbj.org/pub/pdb/validation_reports/bk/8bk8 | HTTPS FTP |
-Related structure data
Related structure data | 16092MC 8bk7C 8bk9C 8bkaC 8bkbC 8bkgC 8bkzC 8bl2C 8bl7C 8blcC 8bldC 8bleC 8blfC 8blyC 8bm0C 8bm1C 8bmdC 8bmoC 8bmtC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 117488.375 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: lacZ, b0344, JW0335 / Production host: Escherichia coli (E. coli) / References: UniProt: P00722, beta-galactosidase |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Beta-galactosidase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | |||||||||||||||||||||||||
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Source (natural) | Organism: Escherichia coli (E. coli) | |||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | |||||||||||||||||||||||||
Buffer solution | pH: 8 / Details: contains Amaranth dye (acid red 27) 32 mM | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 2.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Beta-galactosidase in buffer of Amaranth dye (acid red 27 concentration 32 mM) | |||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1 | |||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Microscopy | Model: JEOL CRYO ARM 300 |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm / Cs: 2.55 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER |
Image recording | Average exposure time: 2.796 sec. / Electron dose: 59 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1409 |
EM imaging optics | Energyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV |
-Processing
Software | Name: PHENIX / Version: 1.19.1_4122: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12887 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||
Refine LS restraints |
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