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- EMDB-44010: INF2 at the Barbed End of F-Actin, Leading WH2 focus refinement -

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Basic information

Entry
Database: EMDB / ID: EMD-44010
TitleINF2 at the Barbed End of F-Actin, Leading WH2 focus refinement
Map data
Sample
  • Complex: Actin
    • Complex: INF2 Dimer
      • Protein or peptide: INF2
    • Complex: Actin Filament
      • Protein or peptide: Actin, alpha skeletal muscle
KeywordsActin / Filament / Elongation / Ends / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / regulation of mitochondrial fission / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle / actin filament bundle assembly / skeletal muscle myofibril ...cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / regulation of mitochondrial fission / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / stress fiber / skeletal muscle fiber development / 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 / 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 speciesOryctolagus cuniculus (rabbit) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.17 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
DepositionMar 9, 2024-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateAug 21, 2024-
Current statusAug 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44010.png

Downloads & links

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Map

FileDownload / File: emd_44010.map.gz / Format: CCP4 / Size: 216 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 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

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: 0.119
Minimum - Maximum-0.17300135 - 0.6317757
Average (Standard dev.)-0.00018714603 (±0.017278116)
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_44010_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44010_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44010_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Actin

EntireName: Actin
Components
  • Complex: Actin
    • Complex: INF2 Dimer
      • Protein or peptide: INF2
    • Complex: Actin Filament
      • Protein or peptide: Actin, alpha skeletal muscle

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Supramolecule #1: Actin

SupramoleculeName: Actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 264 KDa

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Supramolecule #2: INF2 Dimer

SupramoleculeName: INF2 Dimer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
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

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWHH TFYNELRVAP EEHPTLLTEA PLNPKANREK MTQIMFETFN VPAMYVAIQA VLSLYASGRT TGIVLDSGDG VTHNVPIYEG ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWHH TFYNELRVAP EEHPTLLTEA PLNPKANREK MTQIMFETFN VPAMYVAIQA VLSLYASGRT TGIVLDSGDG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSLEK SYELPDGQVI TIGNERFRCP ETLFQPSFIG MESAGIHETT YNSIMKCDID IRKDLYANNV MSGGTTMYPG IADRMQKEIT ALAPSTMKIK IIAPPERKYS VWIGGSILAS LSTFQQMWIT KQEYDEAGPS IVHRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: INF2

MacromoleculeName: INF2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSVKEGAQRK WAALKEKLGP QDSDPTEANL ESADPELCIR LLQMPSVVNY SGLRKRLEGS DGGWMVQFLE QSGLDLLLEA LARLSGRGVA RISDALLQLT CVSCVRAVMN SRQGIEYILS NQGYVRQLSQ ALDTSNVMVK KQVFELLAAL CIYSPEGHVL TLDALDHYKT ...String:
MSVKEGAQRK WAALKEKLGP QDSDPTEANL ESADPELCIR LLQMPSVVNY SGLRKRLEGS DGGWMVQFLE QSGLDLLLEA LARLSGRGVA RISDALLQLT CVSCVRAVMN SRQGIEYILS NQGYVRQLSQ ALDTSNVMVK KQVFELLAAL CIYSPEGHVL TLDALDHYKT VCSQQYRFSI VMNELSGSDN VPYVVTLLSV INAVILGPED LRARTQLRNE FIGLQLLDVL ARLRDLEDAD LLIQLEAFEE AKAEDEEELL RVSGGVDMSS HQEVFASLFH KVSCSPVSAQ LLSVLQGLLH LEPTLRSSQL LWEALESLVN RAVLLASDAQ ECTLEEVVER LLSVKGRPRP SPLVKAHKSV QANLDQSQRG SSPQNTTTPK PSVEGQQPAA AAACEPVDHA QSESILKVSQ PRALEQQAST PPPPPPPPLL PGSSAEPPPP PPPPPLPSVG AKALPTAPPP PPLPGLGAMA PPAPPLPPPL PGSCEFLPPP PPPLPGLGCP PPPPPLLPGM GWGPPPPPPP LLPCTCSPPV AGGMEEVIVA QVDHGLGSAW VPSHRRVNPP TLRMKKLNWQ KLPSNVAREH NSMWASLSSP DAEAVEPDFS SIERLFSFPA AKPKEPTMVA PRARKEPKEI TFLDAKKSLN LNIFLKQFKC SNEEVAAMIR AGDTTKFDVE VLKQLLKLLP EKHEIENLRA FTEERAKLAS ADHFYLLLLA IPCYQLRIEC MLLCEGAAAV LDMVRPKAQL VLAACESLLT SRQLPIFCQL ILRIGNFLNY GSHTGDADGF KISTLLKLTE TKSQQNRVTL LHHVLEEAEK SHPDLLQLPR DLEQPSQAAG INLEIIRSEA SSNLKKLLET ERKVSASVAE VQEQYTERLQ ASISAFRALD ELFEAIEQKQ RELADYLCED AQQLSLEDTF STMKAFRDLF LRALKENKDR KEQAAKAERR KQQLAEEEAR RPRGEDGKPV RKGPGKQEEV CVIDALLADI RKGFQLRKTA RGRGDTDGGS KAASMDPPRA TEPVATSNPA GDPVGSTRCP ASEPGLDATT ASESRGWDLV DAVTPGPQPT LEQLEEGGPR PLERRSSWYV DASDVLTTED PQCPQPLEGA WPVTLGDAQA LKPLKFSSNQ PPAAGSSRQD AKDPTSLLGV LQAEADSTSE GLEDAVHSRG ARPPAAGPGG DEDEDEEDTA PESALDTSLD KSFSEDAVTD SSGSGTLPRA RGRASKGTGK RRKKRPSRSQ EEVPPDSDDN KTKKLCVIQ

UniProtKB: Inverted formin-2

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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 / 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
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: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 35549
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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