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- EMDB-44943: INF2 in the middle of F-Actin, Focus refinement for mobile WH2 -

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Basic information

Entry
Database: EMDB / ID: EMD-44943
TitleINF2 in the middle of F-Actin, Focus refinement for mobile WH2
Map data
Sample
  • Complex: INF2 in the Middle of Actin Filament
    • Complex: INF2
    • Complex: Actin Filament
KeywordsActin / Formin / Filament / Severing / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 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 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction. However, the lack of structures bound to the actin filament ...Humans express 15 formins that play crucial roles in actin-based processes, including 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. Whereas 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, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly whereas DIA1 has potent elongation activity but does not sever. Using cryo-electron microscopy (cryo-EM) we show 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 formin-homology 2 and Wiskott-Aldrich syndrome protein-homology 2 (FH2 and WH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These 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-
UpdateAug 21, 2024-
Current statusAug 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44943.png

Downloads & links

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Map

FileDownload / File: emd_44943.map.gz / Format: CCP4 / Size: 91.1 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 288 pix.
= 311.04 Å		1.08 Å/pix.
x 288 pix.
= 311.04 Å		1.08 Å/pix.
x 288 pix.
= 311.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.126
Minimum - Maximum-0.4049388 - 0.86510026
Average (Standard dev.)0.0016539674 (±0.030031966)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 311.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44943_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44943_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44943_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 : INF2 in the Middle of Actin Filament

EntireName: INF2 in the Middle of Actin Filament
Components
  • Complex: INF2 in the Middle of Actin Filament
    • Complex: INF2
    • Complex: Actin Filament

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Supramolecule #1: INF2 in the Middle of Actin Filament

SupramoleculeName: INF2 in the Middle of Actin Filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: INF2

SupramoleculeName: INF2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
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: #2
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 mMEGTA
1.0 mMDTT
2.0 mMATP
2.0 mMMgCl2
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: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 82395
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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Atomic model buiding 1

Initial model
PDB IDChainDetails

8f8p

source_name: PDB, initial_model_type: experimental model
source_name: AlphaFold, initial_model_type: in silico modelMultimer
RefinementProtocol: RIGID BODY FIT

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