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- PDB-6e18: Crystal structure of Chlamydomonas reinhardtii HAP2 ectodomain pr... -

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Basic information

Entry
Database: PDB / ID: 6.0E+18
TitleCrystal structure of Chlamydomonas reinhardtii HAP2 ectodomain provides structural insights of functional loops in green algae.
ComponentsHapless 2
KeywordsMEMBRANE PROTEIN / Gamete fusion / membrane binding motif / glycoprotein / cystine ladder
Function / homology
Function and homology information


fusion of sperm to egg plasma membrane involved in single fertilization / cell projection membrane / protein insertion into membrane / cytoplasmic vesicle membrane / cytoplasmic vesicle / lipid binding / plasma membrane
Similarity search - Function
Generative cell specific-1/HAP2 domain / Male gamete fusion factor / HAP2/GCS1
Similarity search - Domain/homology
Biological speciesChlamydomonas reinhardtii (plant)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.6 Å
AuthorsBaquero, E. / Legrand, P. / Rey, F.A.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)340371European Union
CitationJournal: Structure / Year: 2019
Title: Species-Specific Functional Regions of the Green Alga Gamete Fusion Protein HAP2 Revealed by Structural Studies.
Authors: Baquero, E. / Fedry, J. / Legrand, P. / Krey, T. / Rey, F.A.
History
DepositionJul 9, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 7, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 28, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 16, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Data collection / Category: chem_comp / pdbx_audit_support
Item: _chem_comp.type / _pdbx_audit_support.country / _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_special_symmetry / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _atom_site_anisotrop.U[1][1] / _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][2] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.U[3][3] / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_auth_atom_id / _atom_site_anisotrop.pdbx_auth_comp_id / _atom_site_anisotrop.pdbx_auth_seq_id / _atom_site_anisotrop.pdbx_label_asym_id / _atom_site_anisotrop.pdbx_label_atom_id / _atom_site_anisotrop.pdbx_label_comp_id / _atom_site_anisotrop.type_symbol / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_special_symmetry.label_asym_id / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag
Revision 2.2Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hapless 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,1246
Polymers61,4331
Non-polymers1,6925
Water1,49583
1
A: Hapless 2
hetero molecules

A: Hapless 2
hetero molecules

A: Hapless 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)189,37318
Polymers184,2983
Non-polymers5,07515
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-y,x-y+1,z1
crystal symmetry operation3_455-x+y-1,-x,z1
Buried area27380 Å2
ΔGint-44 kcal/mol
Surface area63360 Å2
MethodPISA
2
A: Hapless 2
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)378,74536
Polymers368,5966
Non-polymers10,15030
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-y,x-y+1,z1
crystal symmetry operation3_455-x+y-1,-x,z1
crystal symmetry operation10_554-y,-x,-z-1/21
crystal symmetry operation11_454-x+y-1,y,-z-1/21
crystal symmetry operation12_564x,x-y+1,-z-1/21
Buried area56840 Å2
ΔGint-119 kcal/mol
Surface area124650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)125.620, 125.620, 367.880
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322
Components on special symmetry positions
IDModelComponents
11A-1182-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Hapless 2 / HAP2 / Generative cell specific-1


Mass: 61432.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: HAP2, GCS1 / Cell line (production host): Schneider S2 cells / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: A4GRC6

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Sugars , 3 types, 4 molecules

#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Polysaccharide alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1- ...alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 732.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3/a4-b1_a6-d1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{}}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 84 molecules

#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 83 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY

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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: 6.91 Å3/Da / Density % sol: 80 % / Description: Small hexagonal plates
Preparation: The sample used for crystallization was a complex of Chlamydomonas reinhardtii HAP2 ectodomain with an antibody fragment scFv. However we could not reveal the presence of the scFv in our ...Preparation: The sample used for crystallization was a complex of Chlamydomonas reinhardtii HAP2 ectodomain with an antibody fragment scFv. However we could not reveal the presence of the scFv in our electron density maps. There are two possible explanations: 1) The bound scFv was displaced due to crystallization conditions/packing or 2) The scFv is still bound but disordered because is interacting with one of the disordered loops located in the large solvent volumes between the protomers. In this last case, it is expected that the solvent content of the crystal would be lower than the one currently reported
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.2M LiSO4, 0.1M Tris-HCl pH 8.5 and 25% w/v PEG 5000 MME

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.9786 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Feb 4, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9786 Å / Relative weight: 1
ReflectionResolution: 2.6→40.13 Å / Num. obs: 53971 / % possible obs: 99.9 % / Redundancy: 19.6 % / Biso Wilson estimate: 67.34 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.0592 / Rpim(I) all: 0.091 / Rrim(I) all: 0.406 / Net I/σ(I): 8.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
2.6-2.6719.90.2134639190.2444.86321.90399.6
11.62-40.1315.60.0557390.9990.0140.05797.5

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
XDSJan 26, 2018data reduction
Aimless0.6.2data scaling
PHASER2.7.17phasing
BUSTER2.10.3refinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5MF1

5mf1


Resolution: 2.6→38.99 Å / Cor.coef. Fo:Fc: 0.903 / Cor.coef. Fo:Fc free: 0.891 / SU R Cruickshank DPI: 0.277 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.281 / SU Rfree Blow DPI: 0.227 / SU Rfree Cruickshank DPI: 0.227
Details: Due to marked diffraction anisotropy we post-processed the scaled intensities with the STARANISO program that performs an ellipsoidal resolution cut-off. The classical completeness is 70% up ...Details: Due to marked diffraction anisotropy we post-processed the scaled intensities with the STARANISO program that performs an ellipsoidal resolution cut-off. The classical completeness is 70% up to 2.6A resolution, but the "ellipsoidal completeness" is 94.5% up to the same resolution (79% in the last ellipsoidal shell).
RfactorNum. reflection% reflectionSelection details
Rfree0.234 1897 5.04 %RANDOM
Rwork0.214 ---
obs0.215 37628 69.9 %-
Displacement parametersBiso max: 136.28 Å2 / Biso mean: 50.89 Å2 / Biso min: 21.23 Å2
Baniso -1Baniso -2Baniso -3
1-0.7573 Å20 Å20 Å2
2--0.7573 Å20 Å2
3----1.5146 Å2
Refine analyzeLuzzati coordinate error obs: 0.41 Å
Refinement stepCycle: final / Resolution: 2.6→38.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3943 0 111 83 4137
Biso mean--94.96 42.57 -
Num. residues----529
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1393SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes694HARMONIC5
X-RAY DIFFRACTIONt_it4156HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion580SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance1HARMONIC1
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact4480SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d4156HARMONIC20.008
X-RAY DIFFRACTIONt_angle_deg5688HARMONIC21.07
X-RAY DIFFRACTIONt_omega_torsion2.69
X-RAY DIFFRACTIONt_other_torsion17.02
LS refinement shellResolution: 2.6→2.67 Å / Rfactor Rfree error: 0 / Total num. of bins used: 19
RfactorNum. reflection% reflection
Rfree0.2705 19 5.18 %
Rwork0.2452 348 -
all0.2465 367 -
obs--8.97 %
Refinement TLS params.Method: refined / Origin x: -51.3026 Å / Origin y: 26.2073 Å / Origin z: -25.4354 Å
111213212223313233
T-0.1059 Å20.087 Å2-0.0411 Å2--0.0772 Å2-0.0425 Å2---0.0256 Å2
L0.3656 °20.0642 °20.0358 °2-0.4622 °2-0.056 °2--2.0004 °2
S0.0624 Å °0.035 Å °-0.0634 Å °0.0062 Å °0.0854 Å °-0.0718 Å °0.3444 Å °0.4236 Å °-0.1478 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|23 - A|591 A|1001 - A|1008 A|1101 - A|1183 A|1009 - A|1009 }A23 - 591
2X-RAY DIFFRACTION1{ A|23 - A|591 A|1001 - A|1008 A|1101 - A|1183 A|1009 - A|1009 }A1001 - 1008
3X-RAY DIFFRACTION1{ A|23 - A|591 A|1001 - A|1008 A|1101 - A|1183 A|1009 - A|1009 }A1101 - 1183
4X-RAY DIFFRACTION1{ A|23 - A|591 A|1001 - A|1008 A|1101 - A|1183 A|1009 - A|1009 }A1009

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