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- EMDB-42948: Microtubule protofilament reconstruction in CCP5:microtubule clas... -

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Basic information

Entry
Database: EMDB / ID: EMD-42948
TitleMicrotubule protofilament reconstruction in CCP5:microtubule class#1 complex
Map dataProtofilament reconstruction in class 1, b-factor sharpened
Sample
  • Complex: Microtubule protofilament
    • Complex: Microtubule protofilament
Keywordsmicrotubules / cytoskeleton / STRUCTURAL PROTEIN
Biological speciesSus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsChen J / Zehr EA / Gruschus JM / Szyk A / Liu Y / Tanner ME / Tjandra N / Roll-Mecak A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)1ZIANS003163 United States
CitationJournal: Nature / Year: 2024
Title: Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.
Authors: Jiayi Chen / Elena A Zehr / James M Gruschus / Agnieszka Szyk / Yanjie Liu / Martin E Tanner / Nico Tjandra / Antonina Roll-Mecak /
Abstract: Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes. Glutamylation-the addition of branched ...Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function, and mutations in CCPs lead to human disease. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant β-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.
History
DepositionNov 28, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42948.png

Downloads & links

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Map

FileDownload / File: emd_42948.map.gz / Format: CCP4 / Size: 381.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProtofilament reconstruction in class 1, b-factor sharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 464 pix.
= 577.68 Å		1.25 Å/pix.
x 464 pix.
= 577.68 Å		1.25 Å/pix.
x 464 pix.
= 577.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.245 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.0664483 - 0.15862423
Average (Standard dev.)0.00006143514 (±0.0018613524)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions464464464
Spacing464464464
CellA=B=C: 577.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42948_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Protofilament reconstruction in class 1, raw full map

Fileemd_42948_additional_1.map
AnnotationProtofilament reconstruction in class 1, raw full map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Protofilament reconstruction in class 1, DeepEMhancer processed

Fileemd_42948_additional_2.map
AnnotationProtofilament reconstruction in class 1, DeepEMhancer processed
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Protofilament reconstruction in class 1, raw half map

Fileemd_42948_half_map_1.map
AnnotationProtofilament reconstruction in class 1, raw half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Protofilament reconstruction in class 1, raw half map

Fileemd_42948_half_map_2.map
AnnotationProtofilament reconstruction in class 1, raw half 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 : Microtubule protofilament

EntireName: Microtubule protofilament
Components
  • Complex: Microtubule protofilament
    • Complex: Microtubule protofilament

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Supramolecule #1: Microtubule protofilament

SupramoleculeName: Microtubule protofilament / type: complex / ID: 1 / Parent: 0
Details: Protofilament reconstruction of CCP5 decorated microtubules

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Supramolecule #2: Microtubule protofilament

SupramoleculeName: Microtubule protofilament / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Sus scrofa (pig) / Organ: brain

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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

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMKClpotassium chloride
1.0 mMC9H15O6PTCEP
GridModel: Au-flat 1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 45 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 303 K / Instrument: LEICA EM GP
DetailsPig brain microtubules double cycled with GMPCPP and decorated with human CCP5.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 1.651 sec. / Average electron dose: 53.34 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 135000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 162521
Startup modelType of model: OTHER
Details: Synthetic microtubule references were generated in UCSF Chimera.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 239209
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6dpu


Chain - Source name: PDB / Chain - Initial model type: experimental model

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