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- EMDB-21919: 14-protofilament, GMPCPP stabilized microtubule with nucleotide f... -

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Basic information

Entry
Database: EMDB / ID: EMD-21919
Title14-protofilament, GMPCPP stabilized microtubule with nucleotide free kinesin
Map dataReconstruction from a 14-protofilament, GMPCPP-stabilized microtubule fully decorated with kinesin.
Sample
  • Organelle or cellular component: Microtubules stabilized with GMPCPP, fully decorated with kinesin in the nucleotide free state
    • Protein or peptide: alpha-Tubulin
    • Protein or peptide: beta-Tubulin
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDebs GE / Cha M / Sindelar CV
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy.
Authors: Garrett E Debs / Michael Cha / Xueqi Liu / Andrew R Huehn / Charles V Sindelar /
Abstract: Microtubules are tubular polymers with essential roles in numerous cellular activities. Structures of microtubules have been captured at increasing resolution by cryo-EM. However, dynamic properties ...Microtubules are tubular polymers with essential roles in numerous cellular activities. Structures of microtubules have been captured at increasing resolution by cryo-EM. However, dynamic properties of the microtubule are key to its function, and this behavior has proved difficult to characterize at a structural level due to limitations in existing structure determination methods. We developed a high-resolution cryo-EM refinement method that divides an imaged microtubule into its constituent protofilaments, enabling deviations from helicity and other sources of heterogeneity to be quantified and corrected for at the single-subunit level. We demonstrate that this method improves the resolution of microtubule 3D reconstructions and substantially reduces anisotropic blurring artifacts, compared with methods that utilize helical symmetry averaging. Moreover, we identified an unexpected, discrete behavior of the m-loop, which mediates lateral interactions between neighboring protofilaments and acts as a flexible hinge between them. The hinge angle adopts preferred values corresponding to distinct conformations of the m-loop that are incompatible with helical symmetry. These hinge angles fluctuate in a stochastic manner, and perfectly cylindrical microtubule conformations are thus energetically and entropically penalized. The hinge angle can diverge further from helical symmetry at the microtubule seam, generating a subpopulation of highly distorted microtubules. However, the seam-distorted subpopulation disappears in the presence of Taxol, a microtubule stabilizing agent. These observations provide clues into the structural origins of microtubule flexibility and dynamics and highlight the role of structural polymorphism in defining microtubule behavior.
History
DepositionMay 5, 2020-
Header (metadata) releaseMay 20, 2020-
Map releaseMay 20, 2020-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21919.png

Downloads & links

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Map

FileDownload / File: emd_21919.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction from a 14-protofilament, GMPCPP-stabilized microtubule fully decorated with kinesin.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 160 pix.
= 208. Å		1.3 Å/pix.
x 160 pix.
= 208. Å		1.3 Å/pix.
x 160 pix.
= 208. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.0377852 - 0.08832296
Average (Standard dev.)0.00072512304 (±0.0054488447)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 208.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z208.000208.000208.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0380.0880.001

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Supplemental data

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Half map: #1

Fileemd_21919_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_21919_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Microtubules stabilized with GMPCPP, fully decorated with kinesin...

EntireName: Microtubules stabilized with GMPCPP, fully decorated with kinesin in the nucleotide free state
Components
  • Organelle or cellular component: Microtubules stabilized with GMPCPP, fully decorated with kinesin in the nucleotide free state
    • Protein or peptide: alpha-Tubulin
    • Protein or peptide: beta-Tubulin

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Supramolecule #1: Microtubules stabilized with GMPCPP, fully decorated with kinesin...

SupramoleculeName: Microtubules stabilized with GMPCPP, fully decorated with kinesin in the nucleotide free state
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: alpha-Tubulin

MacromoleculeName: alpha-Tubulin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAG KHVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA A NNYARGHY TIGKEIIDLV LDRIRKLADQ CTGLQGFLVF HSFGGGTGSG FT SLLMERL SVDYGKKSKL ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAG KHVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA A NNYARGHY TIGKEIIDLV LDRIRKLADQ CTGLQGFLVF HSFGGGTGSG FT SLLMERL SVDYGKKSKL EFSIYPAPQV STAVVEPYNS ILTTHTTLEH SDC AFMVDN EAIYDICRRN LDIERPTYTN LNRLISQIVS SITASLRFDG ALNV DLTEF QTNLVPYPRI HFPLATYAPV ISAEKAYHEQ LSVAEITNAC FEPAN QMVK CDPRHGKYMA CCLLYRGDVV PKDVNAAIAT IKTKRSIQFV DWCPTG FKV GINYQPPTVV PGGDLAKVQR AVCMLSNTTA IAEAWARLDH KFDLMYA KR AFVHWYVGEG MEEGEFSEAR EDMAALEKDY EEVGVDSVEG EGEEEGEE Y

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Macromolecule #2: beta-Tubulin

MacromoleculeName: beta-Tubulin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKY VPRAILVDLE PGTMDSVRSG PFGQIFRPDN FVFGQSGAGN N WAKGHYTE GAELVDSVLD VVRKESESCD CLQGFQLTHS LGGGTGSGMG TL LISKIRE EYPDRIMNTF ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKY VPRAILVDLE PGTMDSVRSG PFGQIFRPDN FVFGQSGAGN N WAKGHYTE GAELVDSVLD VVRKESESCD CLQGFQLTHS LGGGTGSGMG TL LISKIRE EYPDRIMNTF SVMPSPKVSD TVVEPYNATL SVHQLVENTD ETY SIDNEA LYDICFRTLK LTTPTYGDLN HLVSATMSGV TTCLRFPGQL NADL RKLAV NMVPFPRLHF FMPGFAPLTS RGSQQYRALT VPELTQQMFD SKNMM AACD PRHGRYLTVA AIFRGRMSMK EVDEQMLNVQ NKNSSYFVEW IPNNVK TAV CDIPPRGLKM SATFIGNSTA IQELFKRISE QFTAMFRRKA FLHWYTG EG MDEMEFTEAE SNMNDLVSEY QQYQDATADE QGEFEEEEGE DEA

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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: 6.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 4-20 / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 403424
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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