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- EMDB-28936: Cryo-EM structure of the Tropomodulin-capped pointed end of F-actin -

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Basic information

Entry
Database: EMDB / ID: EMD-28936
TitleCryo-EM structure of the Tropomodulin-capped pointed end of F-actin
Map dataFinal map of the Tropomodulin-bound actin filament pointed end.
Sample
  • Complex: Tropomodulin-capped pointed end of F-actin
    • Complex: Tropomodulin (Tmod)
      • Protein or peptide: Tropomodulin-1
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin cytoskeleton / filament / Tmod / STRUCTURAL PROTEIN
Function / homology
Function and homology information


pointed-end actin filament capping / lens fiber cell development / myofibril assembly / Striated Muscle Contraction / cytoskeletal motor activator activity / tropomyosin binding / cortical cytoskeleton / myofibril / myosin heavy chain binding / mesenchyme migration ...pointed-end actin filament capping / lens fiber cell development / myofibril assembly / Striated Muscle Contraction / cytoskeletal motor activator activity / tropomyosin binding / cortical cytoskeleton / myofibril / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / sarcomere / adult locomotory behavior / filopodium / muscle contraction / actin filament organization / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / actin binding / cytoskeleton / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Tropomodulin / Tropomodulin / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin ...Tropomodulin / Tropomodulin / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / Leucine-rich repeat domain superfamily / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Tropomodulin-1 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human) / Oryctolagus cuniculus (rabbit) / rabbit (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsCarman PJ / Barrie KR / Dominguez R
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM073791 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136511 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL156431 United States
CitationJournal: Science / Year: 2023
Title: Structures of the free and capped ends of the actin filament.
Authors: Peter J Carman / Kyle R Barrie / Grzegorz Rebowski / Roberto Dominguez /
Abstract: The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and ...The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.
History
DepositionNov 22, 2022-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28936.png

Downloads & links

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Map

FileDownload / File: emd_28936.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map of the Tropomodulin-bound actin filament pointed end.
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.172
Minimum - Maximum-0.3879218 - 1.038318
Average (Standard dev.)0.00008147898 (±0.025611743)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened final map of the Tropomodulin-bound actin filament...

Fileemd_28936_additional_1.map
AnnotationSharpened final map of the Tropomodulin-bound actin filament pointed end.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B of the Tropomodulin-bound actin filament pointed end.

Fileemd_28936_half_map_1.map
AnnotationHalf-map B of the Tropomodulin-bound actin filament pointed end.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A of the Tropomodulin-bound actin filament pointed end.

Fileemd_28936_half_map_2.map
AnnotationHalf-map A of the Tropomodulin-bound actin filament pointed end.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tropomodulin-capped pointed end of F-actin

EntireName: Tropomodulin-capped pointed end of F-actin
Components
  • Complex: Tropomodulin-capped pointed end of F-actin
    • Complex: Tropomodulin (Tmod)
      • Protein or peptide: Tropomodulin-1
    • Complex: Actin filamentMicrofilament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Tropomodulin-capped pointed end of F-actin

SupramoleculeName: Tropomodulin-capped pointed end of F-actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Tropomodulin (Tmod)

SupramoleculeName: Tropomodulin (Tmod) / 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
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: 7 / Enantiomer: LEVO
Source (natural)Organism: rabbit (rabbit)
Molecular weightTheoretical: 42.109973 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSS S LEKSYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQ KEITALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #2: Tropomodulin-1

MacromoleculeName: Tropomodulin-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.619078 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSYRRELEKY RDLDEDEILG ALTEEELRTL ENELDELDPD NALLPAGLRQ KDQTTKAPTG PFKREELLDH LEKQAKEFKD REDLVPYTG EKRGKVWVPK QKPLDPVLES VTLEPELEEA LANASDAELC DIAAILGMHT LMSNQQYYQA LSSSSIMNKE G LNSVIKPT ...String:
MSYRRELEKY RDLDEDEILG ALTEEELRTL ENELDELDPD NALLPAGLRQ KDQTTKAPTG PFKREELLDH LEKQAKEFKD REDLVPYTG EKRGKVWVPK QKPLDPVLES VTLEPELEEA LANASDAELC DIAAILGMHT LMSNQQYYQA LSSSSIMNKE G LNSVIKPT QYKPVPDEEP NSTDVEETLE RIKNNDPKLE EVNLNNIRNI PIPTLKAYAE ALKENSYVKK FSIVGTRSND PV AYALAEM LKENKVLKTL NVESNFISGA GILRLVEALP YNTSLVEMKI DNQSQPLGNK VEMEIVSMLE KNATLLKFGY HFT QQGPRL RASNAMMNNN DLVRKRRLAD LTGPIIPKCR SGV

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 7 / 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: 7 / 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

Concentration1.05 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 0 Blot time 2.5 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 44.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1102793 / Details: Topaz particle picking with training
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.0) / Number images used: 25004
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-8f8t:
Cryo-EM structure of the Tropomodulin-capped pointed end of F-actin

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