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- PDB-6zfn: Structure of an inactive E404Q variant of the catalytic domain of... -

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Basic information

Entry
Database: PDB / ID: 6zfn
TitleStructure of an inactive E404Q variant of the catalytic domain of human endo-alpha-mannosidase MANEA in complex with 1-methyl alpha-1,2-mannobiose
ComponentsGlycoprotein endo-alpha-1,2-mannosidase
KeywordsHYDROLASE / Golgi / mannosidase / retaining
Function / homology
Function and homology information


glycoprotein endo-alpha-1,2-mannosidase / glycoprotein endo-alpha-1,2-mannosidase activity / N-glycan trimming and elongation in the cis-Golgi / alpha-mannosidase activity / Golgi membrane / Golgi apparatus
Similarity search - Function
Glycosyl hydrolase family 99 / Glycosyl hydrolase family 99 / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Glycoprotein endo-alpha-1,2-mannosidase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.1 Å
AuthorsSobala, L.F. / Fernandes, P.Z. / Hakki, Z. / Thompson, A.J. / Howe, J.D. / Hill, M. / Zitzmann, N. / Davies, S. / Stamataki, Z. / Butters, T.D. ...Sobala, L.F. / Fernandes, P.Z. / Hakki, Z. / Thompson, A.J. / Howe, J.D. / Hill, M. / Zitzmann, N. / Davies, S. / Stamataki, Z. / Butters, T.D. / Alonzi, D.S. / Williams, S.J. / Davies, G.J.
Funding support United Kingdom, Australia, 4items
OrganizationGrant numberCountry
European Research Council (ERC)322942 United Kingdom
Australian Research Council (ARC)DP120101396 Australia
Australian Research Council (ARC)FT130100103 Australia
Australian Research Council (ARC)DP180101957 Australia
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2020
Title: Structure of human endo-alpha-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.
Authors: Sobala, L.F. / Fernandes, P.Z. / Hakki, Z. / Thompson, A.J. / Howe, J.D. / Hill, M. / Zitzmann, N. / Davies, S. / Stamataki, Z. / Butters, T.D. / Alonzi, D.S. / Williams, S.J. / Davies, G.J.
History
DepositionJun 17, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 2, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jan 20, 2021Group: Database references / Category: pdbx_related_exp_data_set
Revision 1.3Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z ..._atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _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
AAA: Glycoprotein endo-alpha-1,2-mannosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,2343
Polymers44,7821
Non-polymers4522
Water8,611478
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area910 Å2
ΔGint-9 kcal/mol
Surface area15820 Å2
Unit cell
Length a, b, c (Å)42.752, 81.680, 52.994
Angle α, β, γ (deg.)90.000, 92.934, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Glycoprotein endo-alpha-1,2-mannosidase / / hEndo / Mandaselin


Mass: 44782.109 Da / Num. of mol.: 1 / Mutation: E404Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MANEA / Plasmid: pCold-I / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q5SRI9, glycoprotein endo-alpha-1,2-mannosidase
#2: Polysaccharide alpha-D-mannopyranose-(1-2)-methyl alpha-D-mannopyranoside


Type: oligosaccharide / Mass: 356.323 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManp[1Me]a1-OMEGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[a1122h-1a_1-5_1*OC][a1122h-1a_1-5]/1-2/a2-b1WURCSPDB2Glycan 1.1.0
[][methyl]{[(1+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}}LINUCSPDB-CARE
#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 478 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.07 Å3/Da / Density % sol: 40.5 % / Description: hard crystals
Crystal growTemperature: 279 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: 100 mM sodium acetate pH 4.6, 200 mM ammonium sulfate, 12.8% PEG-MME 2000. Protein at 10 mg/ml in 25 mM HEPES pH 7.0, 200 mM NaCl buffer with 2.3 mM GlcMan4OMe (10 x molar ratio). 500 nl ...Details: 100 mM sodium acetate pH 4.6, 200 mM ammonium sulfate, 12.8% PEG-MME 2000. Protein at 10 mg/ml in 25 mM HEPES pH 7.0, 200 mM NaCl buffer with 2.3 mM GlcMan4OMe (10 x molar ratio). 500 nl protein solution and 500 nl reservoir solution.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.97625 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jan 20, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97625 Å / Relative weight: 1
ReflectionResolution: 1.1→52.92 Å / Num. obs: 143298 / % possible obs: 97.6 % / Redundancy: 3.7 % / Biso Wilson estimate: 8.98 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.054 / Rpim(I) all: 0.029 / Rrim(I) all: 0.061 / Net I/σ(I): 11.9
Reflection shellResolution: 1.1→1.12 Å / Redundancy: 2.6 % / Rmerge(I) obs: 0.223 / Mean I/σ(I) obs: 3.3 / Num. unique obs: 5215 / CC1/2: 0.944 / Rpim(I) all: 0.15 / Rrim(I) all: 0.271 / % possible all: 71.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
DIALS1.8.3-g908a951f-releasedata reduction
Aimless0.6.1data scaling
PHASER2.7.17phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6ZDF

6zdf


Resolution: 1.1→52.92 Å / Cor.coef. Fo:Fc: 0.984 / Cor.coef. Fo:Fc free: 0.979 / Cross valid method: FREE R-VALUE / ESU R: 0.023 / ESU R Free: 0.024
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.1279 7225 5.043 %
Rwork0.1063 136038 -
all0.107 --
obs-143263 97.529 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 14.999 Å2
Baniso -1Baniso -2Baniso -3
1--0.257 Å2-0 Å2-0.173 Å2
2--0.472 Å20 Å2
3----0.196 Å2
Refinement stepCycle: LAST / Resolution: 1.1→52.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2922 0 29 478 3429
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0133395
X-RAY DIFFRACTIONr_bond_other_d0.0330.0173040
X-RAY DIFFRACTIONr_angle_refined_deg2.0771.6714687
X-RAY DIFFRACTIONr_angle_other_deg2.5031.5867129
X-RAY DIFFRACTIONr_dihedral_angle_1_deg11.1965.2451
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.65921.487195
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.83615570
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.7911524
X-RAY DIFFRACTIONr_chiral_restr0.1580.2445
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.024058
X-RAY DIFFRACTIONr_gen_planes_other0.0180.02782
X-RAY DIFFRACTIONr_nbd_refined0.2340.2657
X-RAY DIFFRACTIONr_symmetry_nbd_other0.2070.22755
X-RAY DIFFRACTIONr_nbtor_refined0.1930.21581
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0790.21479
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1580.2303
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0870.21
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.2830.236
X-RAY DIFFRACTIONr_nbd_other0.2360.2100
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1720.230
X-RAY DIFFRACTIONr_mcbond_it1.7041.2991534
X-RAY DIFFRACTIONr_mcbond_other1.7051.2991533
X-RAY DIFFRACTIONr_mcangle_it2.3191.9581935
X-RAY DIFFRACTIONr_mcangle_other2.3181.9581936
X-RAY DIFFRACTIONr_scbond_it1.9741.551861
X-RAY DIFFRACTIONr_scbond_other1.9741.551861
X-RAY DIFFRACTIONr_scangle_it2.5542.2292707
X-RAY DIFFRACTIONr_scangle_other2.5542.2292708
X-RAY DIFFRACTIONr_lrange_it3.47416.4854091
X-RAY DIFFRACTIONr_lrange_other3.47216.4814090
X-RAY DIFFRACTIONr_rigid_bond_restr11.68236435
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.1-1.1290.1793990.1547695X-RAY DIFFRACTION74.4344
1.129-1.160.1475290.1339569X-RAY DIFFRACTION96.1165
1.16-1.1930.145050.1219723X-RAY DIFFRACTION99.4361
1.193-1.230.1254960.1159469X-RAY DIFFRACTION99.5505
1.23-1.270.1224690.1079140X-RAY DIFFRACTION99.5236
1.27-1.3150.135070.0978789X-RAY DIFFRACTION99.4544
1.315-1.3640.114320.0878545X-RAY DIFFRACTION99.6337
1.364-1.420.1144110.0848285X-RAY DIFFRACTION99.6219
1.42-1.4830.14050.0757899X-RAY DIFFRACTION99.7238
1.483-1.5560.0923830.0737565X-RAY DIFFRACTION99.6614
1.556-1.640.0973490.0727218X-RAY DIFFRACTION99.7495
1.64-1.7390.0944080.0746737X-RAY DIFFRACTION99.7487
1.739-1.8590.1023540.0796396X-RAY DIFFRACTION99.8964
1.859-2.0080.1063090.0895966X-RAY DIFFRACTION99.9681
2.008-2.20.1243100.15482X-RAY DIFFRACTION99.9482
2.2-2.4590.1262880.0994931X-RAY DIFFRACTION99.8661
2.459-2.8390.132520.1124387X-RAY DIFFRACTION99.8493
2.839-3.4760.1421930.123723X-RAY DIFFRACTION99.7961
3.476-4.9110.1451370.1232914X-RAY DIFFRACTION100
4.911-52.920.25890.221605X-RAY DIFFRACTION99.53

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