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- EMDB-23870: Cryo-EM structure of cortical microtubule from Toxoplasma gondii ... -

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Basic information

Entry
Database: EMDB / ID: EMD-23870
TitleCryo-EM structure of cortical microtubule from Toxoplasma gondii (without TrxL2)
Map dataComposite map of T. gondii cortical microtubule (without TrxL2)
Sample
  • Complex: cortical microtubule with associated MIPs
    • Protein or peptide: SPM1
    • Protein or peptide: alpha-tubulin
    • Protein or peptide: beta-tubulin
    • Protein or peptide: TrxL1
Function / homology
Function and homology information


microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / 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 / Microtubule associated protein SPM1 / Tubulin beta chain / Tubulin alpha chain / PDI family protein
Similarity search - Component
Biological speciesToxoplasma gondii (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWang X / Brown A / Sibley LD / Zhang R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI034036 United States
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM structure of cortical microtubules from human parasite Toxoplasma gondii identifies their microtubule inner proteins.
Authors: Xiangli Wang / Yong Fu / Wandy L Beatty / Meisheng Ma / Alan Brown / L David Sibley / Rui Zhang /
Abstract: In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized ...In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.
History
DepositionApr 18, 2021-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateJun 9, 2021-
Current statusJun 9, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23870.png

Downloads & links

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Map

FileDownload / File: emd_23870.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map of T. gondii cortical microtubule (without TrxL2)
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.15
Minimum - Maximum0.0 - 2.187623
Average (Standard dev.)0.014289515 (±0.08102111)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 438.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0961.0961.096
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z438.400438.400438.400
α/β/γ90.00090.00090.000
start NX/NY/NZ727265
NX/NY/NZ157157169
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean0.0002.1880.014

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Supplemental data

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Additional map: Unsharpened composite map of T. gondii cortical microtubule...

Fileemd_23870_additional_1.map
AnnotationUnsharpened composite map of T. gondii cortical microtubule (without TrxL2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: B-factor sharpened composite map of T. gondii cortical...

Fileemd_23870_additional_2.map
AnnotationB-factor sharpened composite map of T. gondii cortical microtubule (without TrxL2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : cortical microtubule with associated MIPs

EntireName: cortical microtubule with associated MIPs
Components
  • Complex: cortical microtubule with associated MIPs
    • Protein or peptide: SPM1
    • Protein or peptide: alpha-tubulin
    • Protein or peptide: beta-tubulin
    • Protein or peptide: TrxL1

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Supramolecule #1: cortical microtubule with associated MIPs

SupramoleculeName: cortical microtubule with associated MIPs / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Toxoplasma gondii (eukaryote)

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Macromolecule #1: SPM1

MacromoleculeName: SPM1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote)
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 PPVQTVEVKL PPSLPFEGST HYRDEFQVKP LPPATKVTEV KLPPSLPFDA TSMYRSDYVA KSN PICPVS KLPQYPAATY PQNHVFWDPD TKQWY

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Macromolecule #2: alpha-tubulin

MacromoleculeName: alpha-tubulin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote)
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: beta-tubulin

MacromoleculeName: beta-tubulin / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote)
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: TrxL1

MacromoleculeName: TrxL1 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Toxoplasma gondii (eukaryote)
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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds with a blot force of -15.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 2 / Number real images: 9231 / Average exposure time: 9.0 sec. / Average electron dose: 63.7 e/Å2
Details: Images were collected in counting mode, with an exposure rate of 8.5 electrons/pixel/s on the detector camera.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 3.5 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 536132
Details: Cortical MTs were manually picked from the motion-corrected micrographs using the APPION image-processing suite. The selected MT segments were computationally cut into overlapping boxes ...Details: Cortical MTs were manually picked from the motion-corrected micrographs using the APPION image-processing suite. The selected MT segments were computationally cut into overlapping boxes (512x512 pixels) with an 8-nm non-overlapping region (step size) between adjacent boxes (corresponding to the length of a tubulin heterodimer). We refer to these boxed images as 8-nm MT particles.
CTF correctionSoftware: (Name: Gctf (ver. 1.18), RELION (ver. 3.1), cryoSPARC (ver. 2.15))
Details: CTF amplitude correction was performed as part of the 3D reconstruction.
Startup modelType of model: EMDB MAP
Details: We used 13- and 14-protofilament bovine GMPCPP-MTs as the initial models, and performed multi-reference sorting of all the 8-nm MT particles using EMAN1.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: RELION (ver. 3.1), cryoSPARC (ver. 2.15)) / Number images used: 209340
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 300
Projection matching processing - Angular sampling: 3.0 degrees
Software - Name: EMAN (ver. 1.9)
Details: The classification result indicated that T. Gondii cortical MTs are exclusively 13-protofilament MTs.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Details: Next, we performed single-reference refinement using Refine3D in RELION-3 software with global search of alignment parameters. The structure gradually converged to a 13-protofilament MT with ...Details: Next, we performed single-reference refinement using Refine3D in RELION-3 software with global search of alignment parameters. The structure gradually converged to a 13-protofilament MT with 3-start helix and only one seam.
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.1)
Details: To separate the particles with and without TrxL2, we signal-subtracted the tubulin densities from the raw particle images using Particle subtraction in RELION-3, and then performed 3D ...Details: To separate the particles with and without TrxL2, we signal-subtracted the tubulin densities from the raw particle images using Particle subtraction in RELION-3, and then performed 3D classification with a soft-edged cylindrical mask covering only the TrxL2 densities at either site x or y. For each site, the 3D classification was able to produce two clean classes, corresponding to the bound and unbound states of TrxL2.

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Atomic model buiding 1

Initial model(PDB ID:

4fyu

,

6u42

,

3jas

)
DetailsSecondary structure, Ramachandran, and rotamer restraints were applied during refinement. The nonbonded_weight value was set to 500 to reduce the clashes between atoms.
RefinementProtocol: AB INITIO MODEL

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