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- EMDB-16106: Structure of the GroEL-ATP complex plunge-frozen 50 ms after mixi... -

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Basic information

Entry
Database: EMDB / ID: EMD-16106
TitleStructure of the GroEL-ATP complex plunge-frozen 50 ms after mixing with ATP
Map data
Sample
  • Complex: GroEL in complex with ATP
    • Protein or peptide: Chaperonin GroEL
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: POTASSIUM ION
  • Ligand: water
KeywordsGroEL / CHAPERONE
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / cytosol
Similarity search - Function
Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsDhurandhar M / Torino S / Efremov R
Funding supportEuropean Union, Belgium, 3 items
OrganizationGrant numberCountry
European Research Council (ERC)726436European Union
Research Foundation - Flanders (FWO)G0H5916N Belgium
Research Foundation - Flanders (FWO)G054617N Belgium
CitationJournal: 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
DepositionNov 9, 2022-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16106.png

Downloads & links

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Map

FileDownload / File: emd_16106.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 392 pix.
= 376.32 Å		0.96 Å/pix.
x 392 pix.
= 376.32 Å		0.96 Å/pix.
x 392 pix.
= 376.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.019
Minimum - Maximum-0.09035436 - 0.2820614
Average (Standard dev.)0.00019673782 (±0.005716498)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions392392392
Spacing392392392
CellA=B=C: 376.31998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16106_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16106_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16106_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 : GroEL in complex with ATP

EntireName: GroEL in complex with ATP
Components
  • Complex: GroEL in complex with ATP
    • Protein or peptide: Chaperonin GroEL
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: POTASSIUM ION
  • Ligand: water

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Supramolecule #1: GroEL in complex with ATP

SupramoleculeName: GroEL in complex with ATP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: GroEL plunged with 50 ms delay time after mixing with ATP.
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: cytoplasm
Molecular weightTheoretical: 800 KDa

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Macromolecule #1: Chaperonin GroEL

MacromoleculeName: Chaperonin GroEL / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: chaperonin ATPase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 57.391711 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGT TTATVLAQAI ITEGLKAVAA GMNPMDLKRG IDKAVTAAVE ELKALSVPCS DSKAIAQVGT ISANSDETVG K LIAEAMDK ...String:
MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGT TTATVLAQAI ITEGLKAVAA GMNPMDLKRG IDKAVTAAVE ELKALSVPCS DSKAIAQVGT ISANSDETVG K LIAEAMDK VGKEGVITVE DGTGLQDELD VVEGMQFDRG YLSPYFINKP ETGAVELESP FILLADKKIS NIREMLPVLE AV AKAGKPL LIIAEDVEGE ALATLVVNTM RGIVKVAAVK APGFGDRRKA MLQDIATLTG GTVISEEIGM ELEKATLEDL GQA KRVVIN KDTTTIIDGV GEEAAIQGRV AQIRQQIEEA TSDYDREKLQ ERVAKLAGGV AVIKVGAATE VEMKEKKARV EDAL HATRA AVEEGVVAGG GVALIRVASK LADLRGQNED QNVGIKVALR AMEAPLRQIV LNCGEEPSVV ANTVKGGDGN YGYNA ATEE YGNMIDMGIL DPTKVTRSAL QYAASVAGLM ITTECMVTDL PKNDAADLGA AGGMGGMGGM GGMM

UniProtKB: Chaperonin GroEL

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 14 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 14 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 14 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 1298 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMNH2C(CH2OH)3Tris Base
50.0 mMKClPotassium chloride
10.0 mMMgCl2Magnesium chloride
4.0 mMATP
0.2 mMCHAPS
19.0 mMAcid Red 27
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: CONTINUOUS / Support film - #0 - Film thickness: 2 / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: HOLEY / Support film - #1 - Film thickness: 30
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
TemperatureMin: 80.0 K
Specialist opticsEnergy filter - Name: In-column Omega Filter
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 5 / Number real images: 1782 / Average electron dose: 63.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.55 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 38440
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8blc:
Structure of the GroEL-ATP complex plunge-frozen 50 ms after mixing with ATP

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