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- PDB-3upc: A general strategy for the generation of human antibody variable ... -

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Basic information

Entry
Database: PDB / ID: 3upc
TitleA general strategy for the generation of human antibody variable domains with increased aggregation resistance
Componentsheavy chain variable domain
KeywordsIMMUNE SYSTEM / immunoglobulin fold
Function / homology
Function and homology information


CD22 mediated BCR regulation / immunoglobulin complex / Fc epsilon receptor (FCERI) signaling / Classical antibody-mediated complement activation / Initial triggering of complement / FCGR activation / immunoglobulin mediated immune response / Role of phospholipids in phagocytosis / Role of LAT2/NTAL/LAB on calcium mobilization / Scavenging of heme from plasma ...CD22 mediated BCR regulation / immunoglobulin complex / Fc epsilon receptor (FCERI) signaling / Classical antibody-mediated complement activation / Initial triggering of complement / FCGR activation / immunoglobulin mediated immune response / Role of phospholipids in phagocytosis / Role of LAT2/NTAL/LAB on calcium mobilization / Scavenging of heme from plasma / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Regulation of Complement cascade / Cell surface interactions at the vascular wall / FCERI mediated MAPK activation / FCGR3A-mediated phagocytosis / antigen binding / Regulation of actin dynamics for phagocytic cup formation / FCERI mediated NF-kB activation / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / Potential therapeutics for SARS / blood microparticle / immune response / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulins / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / TRIETHYLENE GLYCOL / Immunoglobulin heavy variable 3-23
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.8 Å
AuthorsDudgeon, K. / Rouet, R. / Kokmeijer, I. / Langley, D.B. / Christ, D.
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2012
Title: General strategy for the generation of human antibody variable domains with increased aggregation resistance
Authors: Dudgeon, K. / Rouet, R. / Kokmeijer, I. / Schofield, P. / Stolp, J. / Langley, D. / Stock, D. / Christ, D.
History
DepositionNov 17, 2011Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Jun 20, 2012Provider: repository / Type: Initial release
Revision 1.1Aug 15, 2012Group: Database references
Revision 1.2Nov 8, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.3Nov 1, 2023Group: 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_ncs_dom_lim / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: heavy chain variable domain
B: heavy chain variable domain
C: heavy chain variable domain
D: heavy chain variable domain
E: heavy chain variable domain
F: heavy chain variable domain
G: heavy chain variable domain
H: heavy chain variable domain
I: heavy chain variable domain
J: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)122,42917
Polymers121,11310
Non-polymers1,3167
Water00
1
A: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,3502
Polymers12,1111
Non-polymers2381
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,2622
Polymers12,1111
Non-polymers1501
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,2172
Polymers12,1111
Non-polymers1061
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,3502
Polymers12,1111
Non-polymers2381
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
E: heavy chain variable domain


Theoretical massNumber of molelcules
Total (without water)12,1111
Polymers12,1111
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
6
F: heavy chain variable domain


Theoretical massNumber of molelcules
Total (without water)12,1111
Polymers12,1111
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
7
G: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,3062
Polymers12,1111
Non-polymers1941
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
8
H: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,3062
Polymers12,1111
Non-polymers1941
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
9
I: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,3062
Polymers12,1111
Non-polymers1941
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
10
J: heavy chain variable domain


Theoretical massNumber of molelcules
Total (without water)12,1111
Polymers12,1111
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
11
A: heavy chain variable domain
B: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,6114
Polymers24,2232
Non-polymers3882
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3310 Å2
ΔGint-21 kcal/mol
Surface area10400 Å2
MethodPISA
12
C: heavy chain variable domain
D: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,5674
Polymers24,2232
Non-polymers3442
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3350 Å2
ΔGint-22 kcal/mol
Surface area10520 Å2
MethodPISA
13
E: heavy chain variable domain
I: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,4173
Polymers24,2232
Non-polymers1941
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3290 Å2
ΔGint-21 kcal/mol
Surface area10240 Å2
MethodPISA
14
F: heavy chain variable domain
J: heavy chain variable domain


Theoretical massNumber of molelcules
Total (without water)24,2232
Polymers24,2232
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3350 Å2
ΔGint-22 kcal/mol
Surface area10470 Å2
MethodPISA
15
G: heavy chain variable domain
H: heavy chain variable domain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,6114
Polymers24,2232
Non-polymers3882
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3280 Å2
ΔGint-21 kcal/mol
Surface area10470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)82.610, 143.120, 145.610
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
31C
41D
51E
61F
71G
81H
91I
101J

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: GLN / Beg label comp-ID: GLN / End auth comp-ID: TYR / End label comp-ID: TYR / Refine code: 2 / Auth seq-ID: 3 - 100 / Label seq-ID: 1 - 98

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB
3CC
4DD
5EE
6FF
7GG
8HH
9II
10JJ

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Components

#1: Antibody
heavy chain variable domain


Mass: 12111.343 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pHEN1 / Production host: Escherichia coli (E. coli) / Strain (production host): TG1 / References: UniProt: P01764*PLUS
#2: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400


Mass: 238.278 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#3: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#4: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#5: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM

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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: 3.55 Å3/Da / Density % sol: 65.39 % / Mosaicity: 0.53 °
Crystal growTemperature: 293 K / Method: counter diffusion in crystal harp capillary / pH: 5.5
Details: 20% PEG 3350, 100mM Citrate, pH 5.5, counter diffusion in Crystal Harp capillary, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95369 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Sep 11, 2011
RadiationMonochromator: single crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95369 Å / Relative weight: 1
ReflectionResolution: 2.8→145.61 Å / Num. all: 43312 / Num. obs: 43312 / % possible obs: 100 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2 / Redundancy: 7.2 % / Rsym value: 0.151 / Net I/σ(I): 10.5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) allRmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRrim(I) allRsym valueNet I/σ(I) obs% possible all
2.8-2.957.40.8360.77714605662590.3060.8360.7772.7100
2.95-3.137.40.5390.5011.54323458780.1980.5390.5014.1100
3.13-3.357.30.3170.2952.64079655680.1160.3170.2956.6100
3.35-3.617.30.20.1864.13776151790.0740.20.1869.6100
3.61-3.967.30.1530.1425.43477347900.0560.1530.14211.7100
3.96-4.437.20.0950.0888.43125843450.0350.0950.08816.6100
4.43-5.117.10.1060.0986.52760938930.0390.1060.09818.1100
5.11-6.266.90.1280.1195.52282332890.0480.1280.11915.5100
6.26-8.856.70.060.05512.51732525950.0230.060.05517.599.9
8.85-43.4186.70.0370.03415.91014715160.0140.0370.03421.799.2

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation4 Å19.97 Å
Translation4 Å19.97 Å

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
SCALA3.3.16data scaling
PHASER2.3.0phasing
REFMACrefinement
PDB_EXTRACT3.1data extraction
Blu-Iceicedata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3QOS

3qos
PDB Unreleased entry


Resolution: 2.8→145.61 Å / Cor.coef. Fo:Fc: 0.926 / Cor.coef. Fo:Fc free: 0.892 / WRfactor Rfree: 0.2246 / WRfactor Rwork: 0.1894 / Occupancy max: 1 / Occupancy min: 1 / FOM work R set: 0.8425 / SU B: 22.691 / SU ML: 0.235 / SU R Cruickshank DPI: 0.596 / SU Rfree: 0.3147 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.315 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.247 2180 5 %RANDOM
Rwork0.2093 ---
all0.2112 43273 --
obs0.2112 43273 99.94 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 105.07 Å2 / Biso mean: 40.975 Å2 / Biso min: 3.7 Å2
Baniso -1Baniso -2Baniso -3
1--2.96 Å20 Å20 Å2
2--2.5 Å2-0 Å2
3---0.46 Å2
Refinement stepCycle: LAST / Resolution: 2.8→145.61 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8129 0 88 0 8217
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0218398
X-RAY DIFFRACTIONr_bond_other_d0.0020.025415
X-RAY DIFFRACTIONr_angle_refined_deg1.4521.94311385
X-RAY DIFFRACTIONr_angle_other_deg0.9293.00313024
X-RAY DIFFRACTIONr_dihedral_angle_1_deg10.32251120
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.25423.028327
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.938151087
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.7681545
X-RAY DIFFRACTIONr_chiral_restr0.0940.21210
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.029786
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021931
Refine LS restraints NCS

Dom-ID: 1 / Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

Auth asym-IDNumberTypeRms dev position (Å)Weight position
A573TIGHT POSITIONAL0.040.05
B573TIGHT POSITIONAL0.030.05
C573TIGHT POSITIONAL0.040.05
D573TIGHT POSITIONAL0.040.05
E573TIGHT POSITIONAL0.030.05
F573TIGHT POSITIONAL0.040.05
G573TIGHT POSITIONAL0.040.05
H573TIGHT POSITIONAL0.040.05
I573TIGHT POSITIONAL0.030.05
J573TIGHT POSITIONAL0.030.05
A443MEDIUM POSITIONAL0.040.5
B443MEDIUM POSITIONAL0.040.5
C443MEDIUM POSITIONAL0.040.5
D443MEDIUM POSITIONAL0.040.5
E443MEDIUM POSITIONAL0.030.5
F443MEDIUM POSITIONAL0.040.5
G443MEDIUM POSITIONAL0.040.5
H443MEDIUM POSITIONAL0.040.5
I443MEDIUM POSITIONAL0.030.5
J443MEDIUM POSITIONAL0.030.5
A573TIGHT THERMAL4.560.5
B573TIGHT THERMAL4.350.5
C573TIGHT THERMAL4.420.5
D573TIGHT THERMAL4.230.5
E573TIGHT THERMAL9.510.5
F573TIGHT THERMAL5.020.5
G573TIGHT THERMAL4.680.5
H573TIGHT THERMAL4.390.5
I573TIGHT THERMAL7.820.5
J573TIGHT THERMAL7.190.5
A443MEDIUM THERMAL4.862
B443MEDIUM THERMAL5.692
C443MEDIUM THERMAL5.532
D443MEDIUM THERMAL5.492
E443MEDIUM THERMAL10.412
F443MEDIUM THERMAL6.142
G443MEDIUM THERMAL6.152
H443MEDIUM THERMAL5.282
I443MEDIUM THERMAL9.172
J443MEDIUM THERMAL8.92
LS refinement shellResolution: 2.8→2.873 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.408 153 -
Rwork0.361 2758 -
all-2911 -
obs--100 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
12.67760.8378-0.70175.11-0.42482.2879-0.18930.1576-0.1629-0.34080.09-0.28540.1430.17760.09930.16060.00370.06710.2843-0.05920.167113.2108-37.4001-12.1044
22.3945-0.225-0.56712.4744-1.52683.9076-0.090.11670.2533-0.0838-0.05610.22150.042-0.35110.14610.1126-0.0152-0.03640.2805-0.14780.2643-12.7367-32.4254-5.2978
34.1204-0.1619-1.1463.7419-0.22382.28740.0218-0.06670.1709-0.11870.0244-0.2710.17420.1701-0.04610.12790.0155-0.01190.091-0.05650.25412.4922-20.01679.5937
42.1888-0.5009-0.25824.22391.15425.15770.11680.08840.18840.059-0.10370.3985-0.3868-0.2365-0.01310.14320.0220.00970.0763-0.04810.3352-12.3711-9.464313.4182
54.97683.5637-2.73598.638-2.72765.8816-0.084-0.6724-0.6186-0.4559-0.7054-0.62450.04011.01720.78940.20450.03560.10240.6630.18460.214215.1358-41.782-39.8251
64.954-1.3785-1.08993.50.65754.72250.11830.4661-0.1150.2536-0.42710.26310.1425-0.37910.30880.1248-0.03550.03490.4392-0.04750.0895-10.5864-44.0018-31.0037
73.237-1.3385-0.86832.3158-0.433.4337-0.1734-0.3832-0.00860.20720.29920.21580.1790.0039-0.12570.1608-0.01410.00290.37890.0850.1464-10.5529-38.589-59.8023
83.8079-0.04170.80381.830.34323.94640.0834-0.25410.30330.0865-0.199-0.0186-0.1544-0.03480.11560.1478-0.0335-0.00570.24270.05410.185614.928-31.9042-67.2666
94.75312.1694-0.19526.61991.78063.9920.33630.10550.422-0.46470.1187-0.7055-0.5464-0.0609-0.4550.22870.00190.17070.02290.06440.432213.0621-9.2694-85.1746
104.03130.2103-2.18834.60110.38572.84190.08730.1570.247-0.05590.03150.2482-0.01120.0186-0.11880.2028-0.0054-0.01290.11370.06240.2504-11.4118-20.5283-81.3274
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A3 - 98
2X-RAY DIFFRACTION1B108 - 115
3X-RAY DIFFRACTION2B3 - 98
4X-RAY DIFFRACTION2A108 - 115
5X-RAY DIFFRACTION3C3 - 98
6X-RAY DIFFRACTION3D108 - 115
7X-RAY DIFFRACTION4D3 - 98
8X-RAY DIFFRACTION4C108 - 115
9X-RAY DIFFRACTION5E3 - 98
10X-RAY DIFFRACTION5I108 - 115
11X-RAY DIFFRACTION6I3 - 98
12X-RAY DIFFRACTION6E108 - 115
13X-RAY DIFFRACTION7G3 - 98
14X-RAY DIFFRACTION7H108 - 115
15X-RAY DIFFRACTION8H3 - 98
16X-RAY DIFFRACTION8G108 - 115
17X-RAY DIFFRACTION9J3 - 98
18X-RAY DIFFRACTION9F108 - 115
19X-RAY DIFFRACTION10F3 - 98
20X-RAY DIFFRACTION10J108 - 115

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