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- EMDB-70754: E.coli GroEL/ES in the bullet conformation 300ms after addition of ATP -

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Basic information

Entry
Database: EMDB / ID: EMD-70754
TitleE.coli GroEL/ES in the bullet conformation 300ms after addition of ATP
Map dataSharpened
Sample
  • Complex: GroEL/ES
    • Complex: GroEL
      • Protein or peptide: GroEL
    • Complex: GroES
      • Protein or peptide: GroES
KeywordsGroEL / GroES / Chaperone
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAlexandrescu L / Lander GC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)1F31NS136003-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM154216 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143805 United States
CitationJournal: IUCrJ / Year: 2025
Title: `Mix-it-up': accessible time-resolved cryo-EM on the millisecond timescale.
Authors: Lauren Alexandrescu / William Lessin / Gabriel C Lander /
Abstract: Biological reactions often involve macromolecules that undergo substrate-induced conformational changes in under a second, yet capturing these transient states remains challenging. While high- ...Biological reactions often involve macromolecules that undergo substrate-induced conformational changes in under a second, yet capturing these transient states remains challenging. While high-resolution structural techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) have advanced our mechanistic understanding of protein-substrate interactions, traditional sample-preparation methods are too slow to capture rapid biochemical events. Time-resolved cryo-EM has emerged as a promising approach to visualize structural dynamics on microsecond-to-millisecond timescales, but its widespread adoption has been limited by costly equipment and challenges in achieving rapid mixing, application and vitrification of samples in a reproducible manner. To address these limitations, we developed `Mix-it-up' (MIU), a modified spray device designed for rapid on-grid mixing and vitrification of cryo-EM samples. By manually applying one sample onto the EM grid, blotting and subsequently spraying the second sample, we achieve on-grid mixing with a vitrification delay of as low as ∼120 ms. We demonstrate MIU's time-resolved capabilities through high-resolution structure determination of mixed samples, pH-induced viral capsid contraction and ligand-dependent complex formation. These findings establish MIU as a cost-effective, versatile tool for studying rapid biochemical processes and lay the groundwork for future applications to time-resolved cryo-EM.
History
DepositionMay 20, 2025-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateNov 12, 2025-
Current statusNov 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70754.png

Downloads & links

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Map

FileDownload / File: emd_70754.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 420 pix.
= 394.8 Å		0.94 Å/pix.
x 420 pix.
= 394.8 Å		0.94 Å/pix.
x 420 pix.
= 394.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.94 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0696
Minimum - Maximum-0.5370834 - 0.79380155
Average (Standard dev.)0.000029095358 (±0.022804214)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 394.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened

Fileemd_70754_additional_1.map
AnnotationUnsharpened
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Half map: #2

Fileemd_70754_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_70754_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : GroEL/ES

EntireName: GroEL/ES
Components
  • Complex: GroEL/ES
    • Complex: GroEL
      • Protein or peptide: GroEL
    • Complex: GroES
      • Protein or peptide: GroES

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Supramolecule #1: GroEL/ES

SupramoleculeName: GroEL/ES / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10.39 KDa

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Supramolecule #2: GroEL

SupramoleculeName: GroEL / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: GroES

SupramoleculeName: GroES / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK LIAEAMDKVG ...String:
MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK LIAEAMDKVG KEGVITVEDG TGLQDELDVV EGMQFDRGYL SPYFINKPET GAVELESPFI LLADKKISNI REMLPVLEAV AKAGKPLLII AEDVEGEALA TLVVNTMRGI VKVAAVKAPG FGDRRKAMLQ DIATLTGGTV ISEEIGMELE KATLEDLGQA KRVVINKDTT TIIDGVGEEA AIQGRVAQIR QQIEEATSDY DREKLQERVA KLAGGVAVIK VGAATEVEMK EKKARVEDAL HATRAAVEEG VVAGGGVALI RVASKLADLR GQNEDQNVGI KVALRAMEAP LRQIVLNCGE EPSVVANTVK GGDGNYGYNA ATEEYGNMID MGILDPTKVT RSALQYAASV AGLMITTECM VTDLPKNDAA DLGAAGGMGG MGGMGGMM

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Macromolecule #2: GroES

MacromoleculeName: GroES / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MNIRPLHDRV IVKRKEVETK SAGGIVLTGS AAAKSTRGEV LAVGNGRILE NGEVKPLDVK VGDIVIFNDG YGVKSEKIDN EEVLIMSESD ILAIVEA

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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

Concentration7.22 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
50.0 mMKClPotassium chloride
1.0 mMMgCl2Magnesium chloride
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 14 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 23.998 kPa / Details: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 68 % / Chamber temperature: 296 K
Details: Vitrified at ambient humidity and room temperature using specialized electrospray device, "Mix-it-Up".

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 70.0 K / Max: 77.0 K
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 3665 / Average exposure time: 4.61 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Calibrated defocus max: 3.829 µm / Calibrated defocus min: 0.1 µm / Calibrated magnification: 148936 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 319945
CTF correctionSoftware - Name: cryoSPARC (ver. v4.6.2) / Type: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.6.2) / Number images used: 25430
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.6.2)
Final 3D classificationNumber classes: 50 / Avg.num./class: 3515 / Software - Name: cryoSPARC (ver. v4.6.2)
FSC plot (resolution estimation)

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