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

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Basic information

Entry
Database: PDB / ID: 9b0k
TitleINF2 in the Middle of F-Actin (Down state)
Components
  • Actin, alpha skeletal muscle
  • Inverted formin-2
KeywordsCYTOSOLIC PROTEIN / Actin / Formin / Filament / Severing
Function / homology
Function and homology information


cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / regulation of mitochondrial fission / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril ...cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / regulation of mitochondrial fission / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / small GTPase binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / 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
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, 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 profile. / WH2 domain / 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
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle / Inverted formin-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsPalmer, N.J. / Barrie, K.R. / Dominguez, R.
Funding support United States, 1items
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
DepositionMar 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jun 19, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Aug 21, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
F: Actin, alpha skeletal muscle
G: Inverted formin-2
H: Inverted formin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)350,48920
Polymers347,7808
Non-polymers2,70912
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41387.227 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit)
References: UniProt: P68135, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Protein Inverted formin-2 / HBEBP2-binding protein C


Mass: 49728.199 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INF2, C14orf151, C14orf173 / Production host: Escherichia coli (E. coli) / References: UniProt: Q27J81
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1INF2 in the Middle of Actin FilamentCOMPLEX#1-#20MULTIPLE SOURCES
2INF2COMPLEX#11RECOMBINANT
3Actin FilamentCOMPLEX#21NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Oryctolagus cuniculus (rabbit)9986
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
250 mMNaCl1
31 mMEGTA1
41 mMDTT1
52 mMATP1
62 mMMgCl21
SpecimenConc.: 1.45 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 41926

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPUimage acquisition
4cryoSPARC4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
12cryoSPARC4classification
13cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 289668 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeInitial refinement model-IDSource nameTypeDetails
18f8p

8f8p

8f8p1PDBexperimental model
2AlphaFoldin silico modelMultimer
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00524374
ELECTRON MICROSCOPYf_angle_d0.95233017
ELECTRON MICROSCOPYf_dihedral_angle_d7.9453352
ELECTRON MICROSCOPYf_chiral_restr0.0563700
ELECTRON MICROSCOPYf_plane_restr0.0074239

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