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- EMDB-42973: CCP5 in complex with microtubules class3 -

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Basic information

Entry
Database: EMDB / ID: EMD-42973
TitleCCP5 in complex with microtubules class3
Map dataComposite map of CCP5 class3 in complex with two microtubule protofilaments, stitched from deepEMhancer processed focused CCP5 map and two protofilament maps.
Sample
  • Complex: CCP5 in complex with microtubules
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Cytosolic carboxypeptidase-like protein 5
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: GLUTAMIC ACID
  • Ligand: ZINC ION
Keywordscarboxypeptidase deglutamylation branch glutamate removal microtubule / HYDROLASE / HYDROLASE-SUBSTRATE complex / HYDROLASE-SUBSTRATE / STRUCTURAL PROTEIN complex
Function / homology
Function and homology information


tubulin-glutamate carboxypeptidase / protein deglutamylation / protein side chain deglutamylation / protein branching point deglutamylation / C-terminal protein deglutamylation / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly ...tubulin-glutamate carboxypeptidase / protein deglutamylation / protein side chain deglutamylation / protein branching point deglutamylation / C-terminal protein deglutamylation / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / EML4 and NUDC in mitotic spindle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / metallocarboxypeptidase activity / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / COPI-mediated anterograde transport / intercellular bridge / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / tubulin binding / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / midbody / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / defense response to virus / microtubule / GTPase activity / GTP binding / proteolysis / zinc ion binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Cytosolic carboxypeptidase-like protein 5 catalytic domain / Cytosolic carboxypeptidase, N-terminal / : / Cytosolic carboxypeptidase N-terminal domain / Peptidase family M14 domain profile. / Peptidase M14, carboxypeptidase A / Zinc carboxypeptidase / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site ...Cytosolic carboxypeptidase-like protein 5 catalytic domain / Cytosolic carboxypeptidase, N-terminal / : / Cytosolic carboxypeptidase N-terminal domain / Peptidase family M14 domain profile. / Peptidase M14, carboxypeptidase A / Zinc carboxypeptidase / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 beta chain / Tubulin alpha-1B chain / Cytosolic carboxypeptidase-like protein 5
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human)
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 29, 2023-
Header (metadata) releaseJul 17, 2024-
Map releaseJul 17, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42973.png

Downloads & links

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Map

FileDownload / File: emd_42973.map.gz / Format: CCP4 / Size: 381.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map of CCP5 class3 in complex with two microtubule protofilaments, stitched from deepEMhancer processed focused CCP5 map and two protofilament maps.
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: 0.15
Minimum - Maximum-0.0019626406 - 1.871617
Average (Standard dev.)0.0017691625 (±0.025873613)
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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Additional map: Composite map of CCP5 class3 in complex with...

Fileemd_42973_additional_1.map
AnnotationComposite map of CCP5 class3 in complex with two microtubule protofilaments, stitched from CCP5 focused and two protofilament raw full maps.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CCP5 in complex with microtubules

EntireName: CCP5 in complex with microtubules
Components
  • Complex: CCP5 in complex with microtubules
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Cytosolic carboxypeptidase-like protein 5
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: GLUTAMIC ACID
  • Ligand: ZINC ION

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Supramolecule #1: CCP5 in complex with microtubules

SupramoleculeName: CCP5 in complex with microtubules / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: Composite structure of CCP5:microtubule class#3

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 50.204445 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2
Details: Beta tubulin from porcine brain microtubules, heterogeneous in tail sequence
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 50.377309 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEGEEDEA (UNK)(UNK)E(UNK)(UNK)

UniProtKB: Tubulin beta chain

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Macromolecule #3: Cytosolic carboxypeptidase-like protein 5

MacromoleculeName: Cytosolic carboxypeptidase-like protein 5 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 68.229547 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: NELRCGGLLF SSRFDSGNLA HVEKVESLSS DGEGVGGGAS ALTSGIASSP DYEFNVWTRP DCAETEFENG NRSWFYFSVR GGMPGKLIK INIMNMNKQS KLYSQGMAPF VRTLPTRPRW ERIRDRPTFE MTETQFVLSF VHRFVEGRGA TTFFAFCYPF S YSDCQELL ...String:
NELRCGGLLF SSRFDSGNLA HVEKVESLSS DGEGVGGGAS ALTSGIASSP DYEFNVWTRP DCAETEFENG NRSWFYFSVR GGMPGKLIK INIMNMNKQS KLYSQGMAPF VRTLPTRPRW ERIRDRPTFE MTETQFVLSF VHRFVEGRGA TTFFAFCYPF S YSDCQELL NQLDQRFPEN HPTHSSPLDT IYYHRELLCY SLDGLRVDLL TITSCHGLRE DREPRLEQLF PDTSTPRPFR FA GKRIFFL SSRVHPGETP SSFVFNGFLD FILRPDDPRA QTLRRLFVFK LIPMLNPDGV VRGHYRTDSR GVNLNRQYLK PDA VLHPAI YGAKAVLLYH HVHSRLNSQS SSEHQPSSCL PPDAPVSDLE KANNLQNEAQ CGHSADRHNA EAWKQTEPAE QKLN SVWIM PQQSAGLEES APDTIPPKES GVAYYVDLHG HASKRGCFMY GNSFSDESTQ VENMLYPKLI SLNSAHFDFQ GCNFS EKNM YARDRRDGQS KEGSGRVAIY KASGIIHSYT LACNYNTGRS VNSIPAACHD NGRASPPPPP AFPSRYTVEL FEQVGR AMA IAALDMAECN PWPRIVLSEH SSLTNLRAWM LKHVRNSRGL SS

UniProtKB: Cytosolic carboxypeptidase-like protein 5

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #6: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 6 / Number of copies: 2 / 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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Macromolecule #7: GLUTAMIC ACID

MacromoleculeName: GLUTAMIC ACID / type: ligand / ID: 7
Details: enzymatically linked to the side chain of GLU B 503 of beta-tubulin
Number of copies: 1 / Formula: GLU
Molecular weightTheoretical: 147.129 Da
Chemical component information

ChemComp-GLU:
GLUTAMIC ACID

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.3 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 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 45
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 303 K / Instrument: LEICA EM GP

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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 / Number images used: 80027
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial model
ChainDetailsPDB ID
source_name: Other, initial_model_type: experimental modelcrystal structure of apo CCP5
source_name: PDB, initial_model_type: experimental modelGMPCPP microtubule

6dpu

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-8v4m:
CCP5 in complex with microtubules class3

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