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Yorodumi- PDB-8bm0: Structure of GroEL:GroES-ATP complex plunge frozen 200 ms after r... -
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-Basic information
Entry | Database: PDB / ID: 8bm0 | ||||||||||||
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Title | Structure of GroEL:GroES-ATP complex plunge frozen 200 ms after reaction initiation | ||||||||||||
Components |
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Keywords | CHAPERONE / GroEL | ||||||||||||
Function / homology | Function and homology information GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat / protein-folding chaperone binding / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / metal ion binding / cytosol Similarity search - Function | ||||||||||||
Biological species | Escherichia coli (E. coli) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||||||||
Authors | Dhurandhar, M. / Torino, S. / 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. #1: Journal: Nat Commun / Year: 2023 Title: BtuB TonB-dependent transporters and BtuG surface lipoproteins form stable complexes for vitamin B uptake in gut Bacteroides. Authors: Javier Abellon-Ruiz / Kalyanashis Jana / Augustinas Silale / Andrew M Frey / Arnaud Baslé / Matthias Trost / Ulrich Kleinekathöfer / Bert van den Berg / Abstract: Vitamin B (cobalamin) is required for most human gut microbes, many of which are dependent on scavenging to obtain this vitamin. Since bacterial densities in the gut are extremely high, competition ...Vitamin B (cobalamin) is required for most human gut microbes, many of which are dependent on scavenging to obtain this vitamin. Since bacterial densities in the gut are extremely high, competition for this keystone micronutrient is severe. Contrasting with Enterobacteria, members of the dominant genus Bacteroides often encode several BtuB vitamin B outer membrane transporters together with a conserved array of surface-exposed B-binding lipoproteins. Here we show that the BtuB transporters from Bacteroides thetaiotaomicron form stable, pedal bin-like complexes with surface-exposed BtuG lipoprotein lids, which bind B with high affinities. Closing of the BtuG lid following B capture causes destabilisation of the bound B by a conserved BtuB extracellular loop, causing translocation of the vitamin to BtuB and subsequent transport. We propose that TonB-dependent, lipoprotein-assisted small molecule uptake is a general feature of Bacteroides spp. that is important for the success of this genus in colonising the human gut. #2: 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 | 8bm0.cif.gz | 1.4 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8bm0.ent.gz | 1.2 MB | Display | PDB format |
PDBx/mmJSON format | 8bm0.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bm/8bm0 ftp://data.pdbj.org/pub/pdb/validation_reports/bm/8bm0 | HTTPS FTP |
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-Related structure data
Related structure data | 16116MC 8bk7C 8bk8C 8bk9C 8bkaC 8bkbC 8bkgC 8bkzC 8bl2C 8bl7C 8blcC 8bldC 8bleC 8blfC 8blyC 8bm1C 8bmdC 8bmoC 8bmtC C: citing same article (ref.) M: map data used to model this data |
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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: 57391.711 Da / Num. of mol.: 14 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: groEL, groL, mopA, b4143, JW4103 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6F5, chaperonin ATPase #2: Protein | Mass: 10472.016 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: groES, groS, mopB, b4142, JW4102 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6F9 #3: Chemical | ChemComp-MG / #4: Chemical | ChemComp-K / #5: Chemical | ChemComp-ATP / Has ligand of interest | N | |
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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: GroEL:GroES-ATP complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.95 MDa / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Escherichia coli (E. coli) / Cellular location: cytoplasm | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
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 FIELDBright-field microscopy / Nominal magnification: 60000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm / Cs: 2.55 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER / Temperature (min): 80 K |
Image recording | Electron dose: 63.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
EM imaging optics | Energyfilter name: In-column Omega Filter |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C7 (7 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17592 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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