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- EMDB-70751: Cowpea chlorotic mottle virus in contracted state 700 ms after pH... -

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Basic information

Entry
Database: EMDB / ID: EMD-70751
TitleCowpea chlorotic mottle virus in contracted state 700 ms after pH shift triggered using Mix-it-up
Map data
Sample
  • Virus: Cowpea chlorotic mottle virus
    • Protein or peptide: Cowpea chlorotic mottle virus capsid
Keywordscowpea chlorotic mottle virus / plant / VIRUS
Biological speciesCowpea chlorotic mottle virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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_70751.png

Downloads & links

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Map

FileDownload / File: emd_70751.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 560 pix.
= 526.4 Å		0.94 Å/pix.
x 560 pix.
= 526.4 Å		0.94 Å/pix.
x 560 pix.
= 526.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.94 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0812
Minimum - Maximum-0.12784834 - 0.24476725
Average (Standard dev.)0.00004346152 (±0.012928108)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions560560560
Spacing560560560
CellA=B=C: 526.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_70751_msk_1.map
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Additional map: #1

Fileemd_70751_additional_1.map
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Half map: #2

Fileemd_70751_half_map_1.map
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Half map: #1

Fileemd_70751_half_map_2.map
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Sample components

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Entire : Cowpea chlorotic mottle virus

EntireName: Cowpea chlorotic mottle virus
Components
  • Virus: Cowpea chlorotic mottle virus
    • Protein or peptide: Cowpea chlorotic mottle virus capsid

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Supramolecule #1: Cowpea chlorotic mottle virus

SupramoleculeName: Cowpea chlorotic mottle virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 12303 / Sci species name: Cowpea chlorotic mottle virus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Vigna unguiculata subsp. unguiculata (cowpea) / Strain: No. 5
Molecular weightTheoretical: 3.8 MDa
Virus shellShell ID: 1 / Name: Capsid / Diameter: 290.0 Å / T number (triangulation number): 3

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Macromolecule #1: Cowpea chlorotic mottle virus capsid

MacromoleculeName: Cowpea chlorotic mottle virus capsid / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Cowpea chlorotic mottle virus / Strain: No. 5
SequenceString:
MSTVGTGKLT RAQRRAAARK NKRNTRVVQP VIVEPIASGQ GKAIKAWTGY SVSKWTASCA AAEAKVTSAI TISLPNELSS ERNKQLKVGR VLLWLGLLPS VSGTVKSCVT ETQTTAAASF QVALAVADNS KDVVAAMYPE AFKGITLEQL TADLTIYLYS SAALTEGDVI VHLEVEHVRP TFDDSFTPVY

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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

Concentration20 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Trisaminomethane
50.0 mMNaClSodium chloride
1.0 mMEDTAEthylenediaminetetraacetic acid

Details: Sprayed with low pH buffer: 0.1M NaOAc, 1mM EDTA, pH 3.6
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 14 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 23.998 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 69 % / Chamber temperature: 296 K
Details: Vitrification performed at ambient humidity and room temperature using specialized "Mix-it-Up" electrospray device.

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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: 3050 / Average exposure time: 4.08 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: 3.9410000000000003 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: 68342
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: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.6.2) / Number images used: 6889
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: 1164 / Software - Name: cryoSPARC (ver. v4.6.2)
FSC plot (resolution estimation)

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