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- EMDB-14151: BtubA(R284G,K286D,F287G):BtubB bacterial tubulin M-loop mutant fo... -

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Basic information

Entry
Database: EMDB / ID: EMD-14151
TitleBtubA(R284G,K286D,F287G):BtubB bacterial tubulin M-loop mutant forming a single protofilament (Prosthecobacter dejongeii)
Map dataBtubA(R284G,K286D,F287G):BtubB bacterial tubulin M-loop mutant forming a single protofilament
Sample
  • Complex: BtubAB heterodimer
    • Protein or peptide: Tubulin domain-containing protein
    • Protein or peptide: Tubulin beta
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
KeywordsCytyoskeleton / tubulin-like / cytomotive filaments / CELL CYCLE
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / microtubule / GTP binding
Similarity search - Function
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-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
Similarity search - Domain/homology
Tubulin domain-containing protein / Tubulin beta
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly) / Prosthecobacter dejongeii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsWagstaff J / Planelles-Herrero VJ
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust202754/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)U105184326 United Kingdom
CitationJournal: Sci Adv / Year: 2023
Title: Diverse cytomotive actins and tubulins share a polymerization switch mechanism conferring robust dynamics.
Authors: James Mark Wagstaff / Vicente José Planelles-Herrero / Grigory Sharov / Aisha Alnami / Frank Kozielski / Emmanuel Derivery / Jan Löwe /
Abstract: Protein filaments are used in myriads of ways to organize other molecules within cells. Some filament-forming proteins couple the hydrolysis of nucleotides to their polymerization cycle, thus ...Protein filaments are used in myriads of ways to organize other molecules within cells. Some filament-forming proteins couple the hydrolysis of nucleotides to their polymerization cycle, thus powering the movement of other molecules. These filaments are termed cytomotive. Only members of the actin and tubulin protein superfamilies are known to form cytomotive filaments. We examined the basis of cytomotivity via structural studies of the polymerization cycles of actin and tubulin homologs from across the tree of life. We analyzed published data and performed structural experiments designed to disentangle functional components of these complex filament systems. Our analysis demonstrates the existence of shared subunit polymerization switches among both cytomotive actins and tubulins, i.e., the conformation of subunits switches upon assembly into filaments. These cytomotive switches can explain filament robustness, by enabling the coupling of kinetic and structural polarities required for cytomotive behaviors and by ensuring that single cytomotive filaments do not fall apart.
History
DepositionJan 18, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14151.png

Downloads & links

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Map

FileDownload / File: emd_14151.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBtubA(R284G,K286D,F287G):BtubB bacterial tubulin M-loop mutant forming a single protofilament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 400 pix.
= 422. Å		1.06 Å/pix.
x 400 pix.
= 422. Å		1.06 Å/pix.
x 400 pix.
= 422. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.055 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.423
Minimum - Maximum-2.5044997 - 4.56334
Average (Standard dev.)0.00045691535 (±0.11493309)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 421.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : BtubAB heterodimer

EntireName: BtubAB heterodimer
Components
  • Complex: BtubAB heterodimer
    • Protein or peptide: Tubulin domain-containing protein
    • Protein or peptide: Tubulin beta
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

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Supramolecule #1: BtubAB heterodimer

SupramoleculeName: BtubAB heterodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: M-loop mutant BtubA(R284G,K286D,F287G)
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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Macromolecule #1: Tubulin domain-containing protein

MacromoleculeName: Tubulin domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Prosthecobacter dejongeii (bacteria)
Molecular weightTheoretical: 51.095012 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKVNNTIVVS IGQAGNQIAA SFWKTVCLEH GIDPLTGQTA PGVAPRGNWS SFFSKLGESS SGSYVPRAIM VDLEPSVIDN VKATSGSLF NPANLISRTE GAGGNFAVGY LGAGREVLPE VMSRLDYEID KCDNVGGIIV LHAIGGGTGS GFGALLIESL K EKYGEIPV ...String:
MKVNNTIVVS IGQAGNQIAA SFWKTVCLEH GIDPLTGQTA PGVAPRGNWS SFFSKLGESS SGSYVPRAIM VDLEPSVIDN VKATSGSLF NPANLISRTE GAGGNFAVGY LGAGREVLPE VMSRLDYEID KCDNVGGIIV LHAIGGGTGS GFGALLIESL K EKYGEIPV LSCAVLPSPQ VSSVVTEPYN TVFALNTLRR SADACLIFDN EALFDLAHRK WNIESPTVDD LNLLITEALA GI TASMRFS GFLTVEISLR ELLTNLVPQP SLHFLMCAFA PLTPPDGSDG EELGIEEMIK SLFDNGSVFA ACSPMEGRFL STA VLYRGI MEDKPLADAA LAAMREKLPL TYWIPTAFKI GYVEQPGISH RKSMVLLANN TEIARVLDRI CHNFDKLWQR KAFA NWYLN EGMSEEQINV LRASAQELVQ SYQVAEESGA KAKVQDSAGD TGMRAAAAGV SDDARGSMSL RDLVDRRR

UniProtKB: Tubulin domain-containing protein

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Macromolecule #2: Tubulin beta

MacromoleculeName: Tubulin beta / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Prosthecobacter dejongeii (bacteria)
Molecular weightTheoretical: 46.49757 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MREILSIHVG QCGNQIADSF WRLALREHGL TEAGTLKEGS NAAANSNMEV FFHKVRDGKY VPRAVLVDLE PGVIARIEGG DMSQLFDES SIVRKIPGAA NNWARGYNVE GEKVIDQIMN VIDSAVEKTK GLQGFLMTHS IGGGSGSGLG SLILERLRQA Y PKKRIFTF ...String:
MREILSIHVG QCGNQIADSF WRLALREHGL TEAGTLKEGS NAAANSNMEV FFHKVRDGKY VPRAVLVDLE PGVIARIEGG DMSQLFDES SIVRKIPGAA NNWARGYNVE GEKVIDQIMN VIDSAVEKTK GLQGFLMTHS IGGGSGSGLG SLILERLRQA Y PKKRIFTF SVVPSPLISD SAVEPYNAIL TLQRILDNAD GAVLLDNEAL FRIAKAKLNR SPNYMDLNNI IALIVSSVTA SL RFPGKLN TDLSEFVTNL VPFPGNHFLT ASFAPMRGAG QEGQVRTNFP DLARETFAQD NFTAAIDWQQ GVYLAASALF RGD VKAKDV DENMATIRKS LNYASYMPAS GGLKLGYAET APEGFASSGL ALVNHTGIAA VFERLIAQFD IMFDNHAYTH WYEN AGVSR DMMAKARNQI ATLAQSYRDA S

UniProtKB: Tubulin beta

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Macromolecule #3: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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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

BufferpH: 7.7
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD
VitrificationCryogen name: NITROGEN

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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 / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 6993 / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2btq

Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 290000
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2btq


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7quq:
BtubA(R284G,K286D,F287G):BtubB bacterial tubulin M-loop mutant forming a single protofilament (Prosthecobacter dejongeii)

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