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- EMDB-44045: INF2 in the Middle of F-Actin (Down state) -

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Basic information

Entry
Database: EMDB / ID: EMD-44045
TitleINF2 in the Middle of F-Actin (Down state)
Map data
Sample
  • Complex: INF2 in the Middle of Actin Filament
    • Complex: INF2
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Actin FilamentMicrofilament
      • Protein or peptide: Inverted formin-2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin / Formin / Filament / Severing / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


cytoskeletal motor activator activity / regulation of mitochondrial fission / tropomyosin binding / mesenchyme migration / myosin heavy chain binding / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament ...cytoskeletal motor activator activity / regulation of mitochondrial fission / tropomyosin binding / mesenchyme migration / myosin heavy chain binding / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / small GTPase binding / calcium-dependent protein binding / lamellipodium / actin binding / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / perinuclear region of cytoplasm / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Inverted formin-2 / Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain profile. / Formin, FH2 domain ...Inverted formin-2 / Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain profile. / Formin, FH2 domain / Formin, FH2 domain superfamily / Formin Homology 2 Domain / Formin homology-2 (FH2) domain profile. / Formin Homology 2 Domain / WH2 motif / WH2 domain / WH2 domain profile. / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Actin, alpha skeletal muscle / Inverted formin-2
Similarity search - Component
Biological speciesHomo sapiens (human) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.03 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
DepositionMar 12, 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_44045.png

Downloads & links

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Map

FileDownload / File: emd_44045.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
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: 0.151
Minimum - Maximum-0.28531355 - 1.0412711
Average (Standard dev.)0.0076841684 (±0.03159009)
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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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
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Actin FilamentMicrofilament
      • Protein or peptide: Inverted formin-2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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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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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 41.387227 KDa
SequenceString: TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP ...String:
TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP HAIMRLDLAG RDLTDYLMKI LTERGYSFVT TAEREIVRDI KEKLCYVALD FENEMATAAS SSSLEKSY E LPDGQVITIG NERFRCPETL FQPSFIGMES AGIHETTYNS IMKCDIDIRK DLYANNVMSG GTTMYPGIAD RMQKEITAL APSTMKIKII APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV HRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: Inverted formin-2

MacromoleculeName: Inverted formin-2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 49.728199 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VPSHRRVNPP TLRMKKLNWQ KLPSNVAREH NSMWASLSSP DAEAVEPDFS SIERLFSFPA AKPKEPTMVA PRARKEPKEI TFLDAKKSL NLNIFLKQFK CSNEEVAAMI RAGDTTKFDV EVLKQLLKLL PEKHEIENLR AFTEERAKLA SADHFYLLLL A IPCYQLRI ...String:
VPSHRRVNPP TLRMKKLNWQ KLPSNVAREH NSMWASLSSP DAEAVEPDFS SIERLFSFPA AKPKEPTMVA PRARKEPKEI TFLDAKKSL NLNIFLKQFK CSNEEVAAMI RAGDTTKFDV EVLKQLLKLL PEKHEIENLR AFTEERAKLA SADHFYLLLL A IPCYQLRI ECMLLCEGAA AVLDMVRPKA QLVLAACESL LTSRQLPIFC QLILRIGNFL NYGSHTGDAD GFKISTLLKL TE TKSQQNR VTLLHHVLEE AEKSHPDLLQ LPRDLEQPSQ AAGINLEIIR SEASSNLKKL LETERKVSAS VAEVQEQYTE RLQ ASISAF RALDELFEAI EQKQRELADY LCEDAQQLSL EDTFSTMKAF RDLFLRALKE NKDRKEQAAK AERRKQQLAE EEAR RPRGE DGKPVRKGPG KQEEVCVIDA LLADIRKGFQ LR

UniProtKB: Inverted formin-2

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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 mMNaClSodium chloride
1.0 mMEGTA
1.0 mMDTT
2.0 mMATPAdenosine triphosphate
2.0 mMMgCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 100 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 41926 / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
Final angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 289668

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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
Output model

PDB-9b0k:
INF2 in the Middle of F-Actin (Down state)

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