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- PDB-5jm6: Structure of Chaetomium thermophilum mApe1 -

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Basic information

Entry
Database: PDB / ID: 5jm6
TitleStructure of Chaetomium thermophilum mApe1
ComponentsAminopeptidase-like protein
KeywordsHYDROLASE / aminopeptidase / dodecamer / cvt pathway / selective autophagy
Function / homology
Function and homology information


aminopeptidase activity / metallopeptidase activity / zinc ion binding
Similarity search - Function
Aminopeptidase i, Domain 2 / Aminopeptidase i, Domain 2 / Peptidase M18 / Peptidase M18, domain 2 / Aminopeptidase I zinc metalloprotease (M18) / Zn peptidases / Aminopeptidase / Roll / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Aminopeptidase-like protein
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.758 Å
AuthorsBertipaglia, C. / Jakobi, A.J. / Wilmanns, M. / Sachse, C.
CitationJournal: EMBO Rep / Year: 2016
Title: Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle.
Authors: Chiara Bertipaglia / Sarah Schneider / Arjen J Jakobi / Abul K Tarafder / Yury S Bykov / Andrea Picco / Wanda Kukulski / Jan Kosinski / Wim Jh Hagen / Arvind C Ravichandran / Matthias ...Authors: Chiara Bertipaglia / Sarah Schneider / Arjen J Jakobi / Abul K Tarafder / Yury S Bykov / Andrea Picco / Wanda Kukulski / Jan Kosinski / Wim Jh Hagen / Arvind C Ravichandran / Matthias Wilmanns / Marko Kaksonen / John Ag Briggs / Carsten Sachse /
Abstract: Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles ...Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.
History
DepositionApr 28, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jun 15, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2016Group: Database references
Revision 1.2Jul 13, 2016Group: Database references
Revision 1.3Oct 16, 2019Group: Data collection / Category: reflns / reflns_shell / Item: _reflns.pdbx_CC_half / _reflns_shell.pdbx_CC_half
Revision 1.4Jan 10, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Aminopeptidase-like protein
E: Aminopeptidase-like protein
F: Aminopeptidase-like protein
B: Aminopeptidase-like protein
C: Aminopeptidase-like protein
D: Aminopeptidase-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)339,18818
Polymers338,4036
Non-polymers78512
Water5,026279
1
A: Aminopeptidase-like protein
E: Aminopeptidase-like protein
F: Aminopeptidase-like protein
B: Aminopeptidase-like protein
C: Aminopeptidase-like protein
D: Aminopeptidase-like protein
hetero molecules

A: Aminopeptidase-like protein
E: Aminopeptidase-like protein
F: Aminopeptidase-like protein
B: Aminopeptidase-like protein
C: Aminopeptidase-like protein
D: Aminopeptidase-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)678,37536
Polymers676,80512
Non-polymers1,57024
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555x,-y,-z1
Buried area77300 Å2
ΔGint-1277 kcal/mol
Surface area162550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)121.019, 143.894, 201.315
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121

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Components

#1: Protein
Aminopeptidase-like protein


Mass: 56400.453 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0065300 / Plasmid: pETM33 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIL / References: UniProt: G0SG74
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Formula: Zn
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 279 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.9 Å3/Da / Density % sol: 57.52 % / Description: bipyramidal
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6.6
Details: PROTEIN BUFFER: 2.0-3.0 mg/ml in 50 mM Tris-HCl pH 7.5, 50 mM NaCl; RESERVOIR BUFFER: 100 mM Hepes, pH 6.6, 4 M sodium formate

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.8726 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jun 17, 2013
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8726 Å / Relative weight: 1
ReflectionResolution: 2.75→58.7 Å / Num. obs: 172990 / % possible obs: 98.7 % / Redundancy: 3.1 % / Biso Wilson estimate: 29 Å2 / CC1/2: 0.98 / Rmerge(I) obs: 0.13 / Net I/av σ(I): 7.7 / Net I/σ(I): 0.98
Reflection shellResolution: 2.75→2.8 Å / Redundancy: 3 % / Rmerge(I) obs: 0.77 / Mean I/σ(I) obs: 1.3 / CC1/2: 0.52 / % possible all: 93.5

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Processing

Software
NameVersionClassification
PHENIX(dev_2313: ???)refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3vat

3vat


Resolution: 2.758→49.073 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 0.06 / Phase error: 25.99
RfactorNum. reflection% reflection
Rfree0.2421 8670 5.01 %
Rwork0.2076 --
obs0.2093 172988 98.91 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.758→49.073 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20394 0 12 279 20685
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01420934
X-RAY DIFFRACTIONf_angle_d1.90828416
X-RAY DIFFRACTIONf_dihedral_angle_d14.53212378
X-RAY DIFFRACTIONf_chiral_restr0.0783204
X-RAY DIFFRACTIONf_plane_restr0.013636
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7584-2.78980.36942700.32615107X-RAY DIFFRACTION92
2.7898-2.82260.35932990.30925480X-RAY DIFFRACTION99
2.8226-2.8570.3542720.30975538X-RAY DIFFRACTION100
2.857-2.89310.35142750.29095567X-RAY DIFFRACTION100
2.8931-2.93120.31843140.28745470X-RAY DIFFRACTION100
2.9312-2.97140.33993110.30245485X-RAY DIFFRACTION99
2.9714-3.01380.37252740.29435534X-RAY DIFFRACTION99
3.0138-3.05880.34542980.28985515X-RAY DIFFRACTION100
3.0588-3.10660.31492600.29415487X-RAY DIFFRACTION100
3.1066-3.15750.34972770.27965551X-RAY DIFFRACTION100
3.1575-3.21190.31342860.26795531X-RAY DIFFRACTION100
3.2119-3.27030.29362810.24965551X-RAY DIFFRACTION99
3.2703-3.33320.28983000.2245478X-RAY DIFFRACTION100
3.3332-3.40120.22732850.22155559X-RAY DIFFRACTION100
3.4012-3.47520.24592760.20595509X-RAY DIFFRACTION99
3.4752-3.5560.2382740.20395512X-RAY DIFFRACTION99
3.556-3.64490.21482680.20145532X-RAY DIFFRACTION100
3.6449-3.74340.22793190.19135484X-RAY DIFFRACTION99
3.7434-3.85350.21832900.17845500X-RAY DIFFRACTION99
3.8535-3.97780.18952950.17015499X-RAY DIFFRACTION99
3.9778-4.11990.19513250.17285471X-RAY DIFFRACTION99
4.1199-4.28480.2142400.16175517X-RAY DIFFRACTION99
4.2848-4.47970.19013190.15135491X-RAY DIFFRACTION99
4.4797-4.71570.17682850.15135501X-RAY DIFFRACTION99
4.7157-5.01090.18992970.15035480X-RAY DIFFRACTION99
5.0109-5.39730.21553430.19115429X-RAY DIFFRACTION99
5.3973-5.93970.23612870.18755414X-RAY DIFFRACTION98
5.9397-6.79720.24322690.20385445X-RAY DIFFRACTION98
6.7972-8.55640.22563230.18545362X-RAY DIFFRACTION97
8.5564-49.08070.20452580.20095319X-RAY DIFFRACTION96

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