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- EMDB-44958: INF2 at the Barbed End of F-Actin with Incoming Profilin-Actin, A... -

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Basic information

Entry
Database: EMDB / ID: EMD-44958
TitleINF2 at the Barbed End of F-Actin with Incoming Profilin-Actin, Actin local refinement
Map data
Sample
  • Complex: Actin
    • Complex: INF2 Dimer
    • Complex: Actin Filament
KeywordsActin / Filament / Elongation / Ends / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsPalmer NJ / Barrie KR / Dominguez R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nature / Year: 2024
Title: Mechanisms of actin filament severing and elongation by formins.
Authors: Nicholas J Palmer / Kyle R Barrie / Roberto Dominguez /
Abstract: Humans express fifteen formins, playing crucial roles in actin-based processes, such as cytokinesis, cell motility, and mechanotransduction . However, the lack of structures bound to the actin ...Humans express fifteen formins, playing crucial roles in actin-based processes, such as cytokinesis, cell motility, and mechanotransduction . However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. While formins are known for their ability to nucleate and elongate F-actin , some formins can additionally depolymerize, sever, or bundle F-actin. Two mammalian formins, inverted formin-2 (INF2) and diaphanous-1 (Dia1), exemplify this diversity. INF2 displays potent severing activity but elongates weakly , whereas Dia1 has potent elongation activity but does not sever . Using cryo-electron microscopy (cryo-EM), we reveal five structural states of INF2 and two of Dia1 bound to the middle and barbed end of F-actin. INF2 and Dia1 bind differently to these sites, consistent with their distinct activities. The FH2 and WH2 domains of INF2 are positioned to sever F-actin, whereas Dia1 appears unsuited for severing. Structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.
History
DepositionMay 20, 2024-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44958.png

Downloads & links

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Map

FileDownload / File: emd_44958.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 384 pix.
= 414.72 Å		1.08 Å/pix.
x 384 pix.
= 414.72 Å		1.08 Å/pix.
x 384 pix.
= 414.72 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.126
Minimum - Maximum-0.24594544 - 0.69226575
Average (Standard dev.)-0.000058799797 (±0.019411655)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 414.72003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44958_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_44958_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44958_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Actin

EntireName: Actin
Components
  • Complex: Actin
    • Complex: INF2 Dimer
    • Complex: Actin Filament

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Supramolecule #1: Actin

SupramoleculeName: Actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 264 KDa

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Supramolecule #2: INF2 Dimer

SupramoleculeName: INF2 Dimer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Actin Filament

SupramoleculeName: Actin Filament / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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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

Concentration1.45 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMHEPES
50.0 mMNaCl
1.0 mMEDTA
1.0 mMDTT
0.05 %Thesit
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 100
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 41926 / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 52040
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
Final angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
FSC plot (resolution estimation)

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