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- EMDB-26018: The symmetry-released subpellicular microtubule map from detergen... -

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Basic information

Entry
Database: EMDB / ID: EMD-26018
TitleThe symmetry-released subpellicular microtubule map from detergent-extracted Toxoplasma cells
Map data
Sample
  • Organelle or cellular component: microtubule complex of tubulin and intra-luminal proteins
    • Protein or peptide: Microtubule associated protein SPM1
    • Protein or peptide: Tubulin alpha chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: PDI family protein
    • Protein or peptide: PDI family protein
Function / homology
Function and homology information


protein-disulfide reductase / protein-disulfide reductase (NAD(P)H) activity / microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule binding / microtubule / hydrolase activity / GTPase activity / GTP binding ...protein-disulfide reductase / protein-disulfide reductase (NAD(P)H) activity / microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule binding / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Stabilizer of axonemal microtubules 1/2 / Thioredoxin-like / Thioredoxin-like fold / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain ...Stabilizer of axonemal microtubules 1/2 / Thioredoxin-like / Thioredoxin-like fold / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / Thioredoxin-like superfamily
Similarity search - Domain/homology
PDI family protein / PDI family protein / Tubulin beta chain / Tubulin alpha chain / Microtubule associated protein SPM1
Similarity search - Component
Biological speciesToxoplasma gondii (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 8.4 Å
AuthorsSun SY / Pintilie GD / Chen M
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01GM121203 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R21MH125285-01A1 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Cryo-ET of parasites gives subnanometer insight into tubulin-based structures.
Authors: Stella Y Sun / Li-Av Segev-Zarko / Muyuan Chen / Grigore D Pintilie / Michael F Schmid / Steven J Ludtke / John C Boothroyd / Wah Chiu /
Abstract: Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in , an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing ...Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in , an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.
History
DepositionJan 21, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26018.png

Downloads & links

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Map

FileDownload / File: emd_26018.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.0715 Å
Density
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-8.372811 - 10.003788
Average (Standard dev.)0.0148649495 (±0.8915399)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 447.444 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : microtubule complex of tubulin and intra-luminal proteins

EntireName: microtubule complex of tubulin and intra-luminal proteins
Components
  • Organelle or cellular component: microtubule complex of tubulin and intra-luminal proteins
    • Protein or peptide: Microtubule associated protein SPM1
    • Protein or peptide: Tubulin alpha chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: PDI family protein
    • Protein or peptide: PDI family protein

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Supramolecule #1: microtubule complex of tubulin and intra-luminal proteins

SupramoleculeName: microtubule complex of tubulin and intra-luminal proteins
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: ME49

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Macromolecule #1: Microtubule associated protein SPM1

MacromoleculeName: Microtubule associated protein SPM1 / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: ATCC 50611 / Me49
Molecular weightTheoretical: 38.817699 KDa
SequenceString: MSGGNSNTPK KLPSEEGSDY GYPQKPQKYL PKSEQAEPDY SACCKGNDAY KGASHGTVQF SHPEEAQKYA GAAAGAETIQ RGRERVAAD RQPRAAGDVP ARRLHLSDVD EAHRGQSPSR HPGYCVEELC TCGMHKCIPS RAPVPFTGST QYRQEFVPKP L PPPTQVSQ ...String:
MSGGNSNTPK KLPSEEGSDY GYPQKPQKYL PKSEQAEPDY SACCKGNDAY KGASHGTVQF SHPEEAQKYA GAAAGAETIQ RGRERVAAD RQPRAAGDVP ARRLHLSDVD EAHRGQSPSR HPGYCVEELC TCGMHKCIPS RAPVPFTGST QYRQEFVPKP L PPPTQVSQ VTLPPSLPFE AESSYRTEFV AKPLPPPAKF SEVKLPPTLP FHGESAYRTD YVPKPLPEVA KPVEVKLPPT LP FNAQSCY RSEYVAKPLP PPVQTAEVKL PPSLPFEGST HYRDEFQVKP LPPATKVTEV KLPPSLPFDA TSMYRSDYVA KSN PICPVS KLPQYPAATY PQNHVFWDPD TKQWY

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Macromolecule #2: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 2 / Number of copies: 26 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: ME49
Molecular weightTheoretical: 50.166645 KDa
SequenceString: MREVISIHVG QAGIQIGNAC WELFCLEHGI QPDGQMPSDK TIGGGDDAFN TFFSETGAGK HVPRCVFLDL EPTVVDEVRT GTYRHLFHP EQLISGKEDA ANNFARGHYT IGKEIVDLSL DRIRKLADNC TGLQGFLMFN AVGGGTGSGL GCLLLERLSV D YGKKSKLN ...String:
MREVISIHVG QAGIQIGNAC WELFCLEHGI QPDGQMPSDK TIGGGDDAFN TFFSETGAGK HVPRCVFLDL EPTVVDEVRT GTYRHLFHP EQLISGKEDA ANNFARGHYT IGKEIVDLSL DRIRKLADNC TGLQGFLMFN AVGGGTGSGL GCLLLERLSV D YGKKSKLN FCSWPSPQVS TAVVEPYNSV LSTHSLLEHT DVAVMLDNEA IYDICRRNLD IERPTYTNLN RLIAQVISSL TA SLRFDGA LNVDVTEFQT NLVPYPRIHF MLSSYAPIIS AEKAYHEQLS VAEITNSAFE PASMMAKCDP RHGKYMACCL MYR GDVVPK DVNAAVATIK TKRTIQFVDW CPTGFKCGIN YQPPTVVPGG DLAKVMRAVC MISNSTAIAE VFSRMDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDL AALEKDYEEV GIETAEGEGE EEGYGDEY

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Macromolecule #3: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 3 / Number of copies: 26 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: ATCC 50611 / Me49
Molecular weightTheoretical: 50.119121 KDa
SequenceString: MREIVHVQGG QCGNQIGAKF WEVISDEHGI DPTGTYCGDS DLQLERINVF YNEATGGRFV PRAILMDLEP GTMDSVRAGP FGQLFRPDN FVFGQTGAGN NWAKGHYTEG AELIDSVLDV VRKEAEGCDC LQGFQITHSL GGGTGSGMGT LLISKVREEY P DRIMETFS ...String:
MREIVHVQGG QCGNQIGAKF WEVISDEHGI DPTGTYCGDS DLQLERINVF YNEATGGRFV PRAILMDLEP GTMDSVRAGP FGQLFRPDN FVFGQTGAGN NWAKGHYTEG AELIDSVLDV VRKEAEGCDC LQGFQITHSL GGGTGSGMGT LLISKVREEY P DRIMETFS VFPSPKVSDT VVEPYNATLS VHQLVENADE VQVIDNEALY DICFRTLKLT TPTYGDLNHL VSAAMSGVTC CL RFPGQLN SDLRKLAVNL IPFPRLHFFL IGFAPLTSRG SQQYRALSVP ELTQQMFDAK NMMCASDPRH GRYLTASAMF RGR MSTKEV DEQMLNVQNK NSSYFVEWIP NNMKSSVCDI PPKGLKMSVT FVGNSTAIQE MFKRVSDQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATAEEEGE FDEEEGEMGA EEGA

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Macromolecule #4: PDI family protein

MacromoleculeName: PDI family protein / type: protein_or_peptide / ID: 4 / Number of copies: 20 / Enantiomer: LEVO / EC number: protein-disulfide reductase
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: ATCC 50611 / Me49
Molecular weightTheoretical: 24.939332 KDa
SequenceString: MSQPVFASPL NVEKRRLNEE RALMQAQKAG GEGVNIQLPP NYGDMDLILF PEGSLKNSNN TVIPQSHLKG KSVALYFADG ADPKCASLL PFLLNYYRTM NEGGANQKIE IIFVSLDRDR EAFESHRAHM PWLSIDLENP LTEILKRHFR VMKEYEVPTY G YGSRTGVP ...String:
MSQPVFASPL NVEKRRLNEE RALMQAQKAG GEGVNIQLPP NYGDMDLILF PEGSLKNSNN TVIPQSHLKG KSVALYFADG ADPKCASLL PFLLNYYRTM NEGGANQKIE IIFVSLDRDR EAFESHRAHM PWLSIDLENP LTEILKRHFR VMKEYEVPTY G YGSRTGVP SVIVIGSDGR EAQFLPICSG LEEGDRALLR WDWRNTKFAS DQFHVRPTLL EQ

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Macromolecule #5: PDI family protein

MacromoleculeName: PDI family protein / type: protein_or_peptide / ID: 5 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: ATCC 50611 / Me49
Molecular weightTheoretical: 21.687934 KDa
SequenceString:
MLAADCFFGP DVVKRTQQGN YVPVRPDHFA GVSVALFFAK AGHSKCAQIV PVVRQFYKTT NFSGEKAVIE IIYVSLDKDE QDFERVRAL MPWCSVEYKS CLRKKLIERY RVPNGELAFG TVRIPSTAIP LLIVIGPNGE EAGRMNFQQS DEFVLQRWDY R FNKWPGSA QRLRTLNDAT DPWKKRLPQN V

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 7.2 / Details: Phosphate Buffered Saline
GridModel: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 0.9 sec. / Average electron dose: 4.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 90 / Number images used: 39122
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 / Number subtomograms used: 39122

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Atomic model buiding 1

Initial modelPDB ID:

7miz

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7tnq:
The symmetry-released subpellicular microtubule map from detergent-extracted Toxoplasma cells

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