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- PDB-6tl0: Solution structure and 1H, 13C and 15N chemical shift assignments... -

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Basic information

Entry
Database: PDB / ID: 6tl0
TitleSolution structure and 1H, 13C and 15N chemical shift assignments for the complex of VPS29 with VARP 687-747
Components
  • Ankyrin repeat domain-containing protein 27
  • Vacuolar protein sorting-associated protein 29
KeywordsINTRACELLULAR VESICLE TRAFFICKING / VARP / retromer / NMR complex structure / Zinc finger / endosome
Function / homology
Function and homology information


negative regulation of SNARE complex assembly / WNT ligand biogenesis and trafficking / tubular endosome / retromer, cargo-selective complex / endosome to melanosome transport / Golgi to vacuole transport / retromer complex / early endosome to late endosome transport / positive regulation of dendrite morphogenesis / RAB GEFs exchange GTP for GDP on RABs ...negative regulation of SNARE complex assembly / WNT ligand biogenesis and trafficking / tubular endosome / retromer, cargo-selective complex / endosome to melanosome transport / Golgi to vacuole transport / retromer complex / early endosome to late endosome transport / positive regulation of dendrite morphogenesis / RAB GEFs exchange GTP for GDP on RABs / endocytic recycling / retrograde transport, endosome to Golgi / transport vesicle / neuron projection morphogenesis / GTPase activator activity / cytoplasmic vesicle membrane / guanyl-nucleotide exchange factor activity / SNARE binding / positive regulation of neuron projection development / small GTPase binding / melanosome / late endosome / protein transport / early endosome / lysosome / neuron projection / endosome membrane / intracellular membrane-bounded organelle / metal ion binding / membrane / plasma membrane / cytosol
Similarity search - Function
: / VPS9 domain / VPS9 domain superfamily / Vacuolar sorting protein 9 (VPS9) domain / VPS9 domain profile. / Domain present in VPS9 / Vacuolar protein sorting-associated protein 29 / Phosphodiesterase MJ0936/Vps29 / Calcineurin-like phosphoesterase domain, lpxH-type / Calcineurin-like phosphoesterase superfamily domain ...: / VPS9 domain / VPS9 domain superfamily / Vacuolar sorting protein 9 (VPS9) domain / VPS9 domain profile. / Domain present in VPS9 / Vacuolar protein sorting-associated protein 29 / Phosphodiesterase MJ0936/Vps29 / Calcineurin-like phosphoesterase domain, lpxH-type / Calcineurin-like phosphoesterase superfamily domain / Metallo-dependent phosphatase-like / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
Ankyrin repeat domain-containing protein 27 / Vacuolar protein sorting-associated protein 29
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsOwen, D.J. / Neuhaus, D. / Yang, J.-C. / Crawley-Snowdon, H.
Funding support United Kingdom, Canada, 4items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)U105178934 United Kingdom
Wellcome Trust090909/Z/09/Z United Kingdom
Natural Sciences and Engineering Research Council (NSERC, Canada)RES0043758 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RES0046091 Canada
CitationJournal: Nat Commun / Year: 2020
Title: Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29.
Authors: Crawley-Snowdon, H. / Yang, J.C. / Zaccai, N.R. / Davis, L.J. / Wartosch, L. / Herman, E.K. / Bright, N.A. / Swarbrick, J.S. / Collins, B.M. / Jackson, L.P. / Seaman, M.N.J. / Luzio, J.P. / ...Authors: Crawley-Snowdon, H. / Yang, J.C. / Zaccai, N.R. / Davis, L.J. / Wartosch, L. / Herman, E.K. / Bright, N.A. / Swarbrick, J.S. / Collins, B.M. / Jackson, L.P. / Seaman, M.N.J. / Luzio, J.P. / Dacks, J.B. / Neuhaus, D. / Owen, D.J.
History
DepositionNov 29, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Vacuolar protein sorting-associated protein 29
B: Ankyrin repeat domain-containing protein 27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,0123
Polymers27,9472
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area660 Å2
ΔGint-5 kcal/mol
Surface area16080 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)25 / 50structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Vacuolar protein sorting-associated protein 29 / Vesicle protein sorting 29


Mass: 21626.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Vps29 / Plasmid: pGEXVPS29 / Details (production host): cDNA Cloned EcoRI into pGEX4T1 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 pLysS / References: UniProt: Q9QZ88
#2: Protein Ankyrin repeat domain-containing protein 27 / VPS9 domain-containing protein


Mass: 6319.927 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANKRD27, PP12899 / Plasmid: pGEXVARPfingernail2 / Details (production host): cDNA clone BamHI/NotI pGEX6P1 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): DE3 pLysS / References: UniProt: Q96NW4
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic32D 1H-15N HSQC
121isotropic32D 1H-13C HSQC aliphatic
131isotropic32D 1H-13C HSQC aromatic
141isotropic32D 1H-13C HSQC aliphatic constant-time
151isotropic32D 1H-1H NOESY (150ms mixing time) filtered to reject 13C-1 and 15N-coupled 1H in F1 and to accept 13C- and 15N-coupled 1H in F2
1151isotropic33D HNCA
1141isotropic33D HN(CO)CA
1131isotropic33D CBCANH
1121isotropic33D CBCA(CO)NH
1111isotropic33D HBHA(CO)NH
1101isotropic33D (H)CCH-TOCSY [1H-13C-1H]
191isotropic33D (H)CCH-TOCSY [13C-13C-1H]
181isotropic33D 15N NOESY-HSQC (150ms mixing time)
171isotropic33D 13C NOESY-HSQC aliphatic (150ms mixing time)
161isotropic33D 13C NOESY-HSQC aromatic (150ms mixing time)
1192isotropic12D 1H-13C HSQC aliphatic
1182isotropic12D 1H-13C HSQC aromatic
1172isotropic12D 1H-13C HSQC aliphatic constant-time
1162isotropic12D 1H-1H NOESY (150ms mixing time) filtered to reject 13C-1 and 15N-coupled 1H in F1 and to accept 13C- and 15N-coupled 1H in F2
1202isotropic12D 1H-1H NOESY (70ms mixing time) filtered to reject 13C-1 and 15N-coupled 1H in F1 and to accept 13C- and 15N-coupled 1H in F2
1292isotropic33D (H)CCH-TOCSY [13C-13C-1H]
1282isotropic13D 13C NOESY-HSQC (50ms mixing time) aliphatic
1272isotropic13D 13C NOESY-HSQC (120ms mixing time) aliphatic
1262isotropic13D 13C NOESY-HSQC (50ms mixing time) aromatic
1252isotropic13D 13C NOESY-HSQC (120ms mixing time) aromatic
1242isotropic13D 13C NOESY-HSQC (50ms mixing time) aliphatic, filtered to reject 13C-1 and 15N-coupled 1H in F1
1232isotropic13D 13C NOESY-HSQC (120ms mixing time) aliphatic, filtered to reject 13C-1 and 15N-coupled 1H in F1
1222isotropic13D 13C NOESY-HSQC (50ms mixing time) aromatic, filtered to reject 13C-1 and 15N-coupled 1H in F1
1212isotropic13D 13C NOESY-HSQC (120ms mixing time) aromatic, filtered to reject 13C-1 and 15N-coupled 1H in F1
1303isotropic32D 1H-15N HSQC
2313isotropic32D 1H-15N HSQC
3323isotropic32D 1H-15N HSQC
1333isotropic32D 1H-13C HSQC
1343isotropic33D CBCA(CO)NH
1353isotropic33D HNCA
1363isotropic33D HN(CO)CA
1373isotropic33D HBHA(CO)NH
1383isotropic33D HNHA
1393isotropic33D (H)CCH-TOCSY [1H-13C-1H]
1403isotropic33D (H)CCH-TOCSY [13C-13C-1H]
1413isotropic33D 1H-15N NOESY-HSQC (70ms mixing time)
1423isotropic33D 1H-15N NOESY-HSQC (150ms mixing time)
2433isotropic33D 1H-15N NOESY-HSQC (50ms mixing time)
3443isotropic13D 1H-15N NOESY-HSQC (50ms mixing time)
1453isotropic33D 1H-13C NOESY (70ms mixing time) aliphatic
1464isotropic12D 1H-13C HSQC aliphatic
1474isotropic12D 1H-13C HSQC aromatic
1484isotropic33D (H)CCH-TOCSY [1H-13C-1H]
1494isotropic33D (H)CCH-TOCSY [13C-13C-1H]
1504isotropic33D 13C NOESY-HSQC aliphatic (50ms mixing time)
1514isotropic13D 13C NOESY-HSQC aliphatic (70ms mixing time)
1524isotropic13D 13C NOESY-HSQC aliphatic (120ms mixing time)
1534isotropic33D 13C NOESY-HSQC aromatic (70ms mixing time)
1542isotropic12D 1H-13C HSQC aromatic constant-time

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution10.45 mM [U-98% 13C; U-98% 15N] VPS29, 0.45 mM VARP 692-746, 20 mM [U-2H] TRIS, 200 mM sodium chloride, 1 mM [U-2H] DTT, 95% H2O/5% D2OH2O sample of 15N,13C-labelled VSP29 with natural abundance VARP 692-746 in 1:1 ratioH2O_sample_labelled_VPS29_unlabelled_VARP95% H2O/5% D2O
solution20.45 mM [U-98% 13C; U-98% 15N] VPS29, 0.45 mM VARP 692-746, 20 mM [U-2H] TRIS, 200 mM sodium chloride, 1 mM [U-2H] DTT, 100% D2OD2O sample of 15N,13C-labelled VSP29 with natural abundance VARP 692-746 in 1:1 ratioD2O_sample_labelled_VPS29_unlabelled_VARP100% D2O
solution30.45 mM VPS29, 0.45 mM [U-98% 13C; U-98% 15N] VARP 692-746, 20 mM [U-2H] TRIS, 200 mM sodium chloride, 1 mM [U-2H] DTT, 95% H2O/5% D2OH2O sample of natural abundance VSP29 with 15N,13C-labelled VARP 692-746 in 1:1 ratioH2O_sample_unlabelled_VPS29_labelled_VARP95% H2O/5% D2O
solution40.45 mM VPS29, 0.45 mM [U-98% 13C; U-98% 15N] VARP 692-746, 20 mM [U-2H] TRIS, 200 mM sodium chloride, 1 mM [U-2H] DTT, 100% D2OD2O sample of natural abundance VSP29 with 15N,13C-labelled VARP 692-746 in 1:1 ratioD2O_sample_unlabelled_VPS29_labelled_VARP100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.45 mMVPS29[U-98% 13C; U-98% 15N]1
0.45 mMVARP 692-746natural abundance1
20 mMTRIS[U-2H]1
200 mMsodium chloridenatural abundance1
1 mMDTT[U-2H]1
0.45 mMVPS29[U-98% 13C; U-98% 15N]2
0.45 mMVARP 692-746natural abundance2
20 mMTRIS[U-2H]2
200 mMsodium chloridenatural abundance2
1 mMDTT[U-2H]2
0.45 mMVPS29natural abundance3
0.45 mMVARP 692-746[U-98% 13C; U-98% 15N]3
20 mMTRIS[U-2H]3
200 mMsodium chloridenatural abundance3
1 mMDTT[U-2H]3
0.45 mMVPS29natural abundance4
0.45 mMVARP 692-746[U-98% 13C; U-98% 15N]4
20 mMTRIS[U-2H]4
200 mMsodium chloridenatural abundance4
1 mMDTT[U-2H]4
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)
1200 mMconditions_171 atm298 K
2200 mMconditions_271 atm283 K
3200 mMconditions_371 atm275 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIIBrukerAVANCE III8001
Bruker AVANCEBrukerAVANCE6003

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin3.5Bruker Biospinprocessing
CcpNmr Analysis2.4.2CCPNchemical shift assignment
X-PLOR NIH2.28Schwieters, Kuszewski, Tjandra and Clorestructure calculation
RefinementMethod: simulated annealing / Software ordinal: 3
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 50 / Conformers submitted total number: 25

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