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- PDB-7p76: Re-engineered 2-deoxy-D-ribose-5-phosphate aldolase catalysing as... -

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Basic information

Entry
Database: PDB / ID: 7p76
TitleRe-engineered 2-deoxy-D-ribose-5-phosphate aldolase catalysing asymmetric Michael addition reactions, Schiff base complex with cinnamaldehyde
ComponentsDeoxyribose-phosphate aldolase
KeywordsLYASE / Protein engineering / directed evolution / aldolase / carboligase / michael addition / Schiff base / cinnamaldehyde / carbon-carbon lyase
Function / homology
Function and homology information


deoxyribose phosphate catabolic process / deoxyribose-phosphate aldolase / deoxyribose-phosphate aldolase activity / deoxyribonucleotide catabolic process / carbohydrate catabolic process / cytoplasm
Similarity search - Function
Deoxyribose-phosphate aldolase type II / Deoxyribose-phosphate aldolase / DeoC/LacD family aldolase / DeoC/FbaB/LacD aldolase / DeoC/LacD family aldolase / Aldolase-type TIM barrel
Similarity search - Domain/homology
(2E)-3-phenylprop-2-enal / Deoxyribose-phosphate aldolase
Similarity search - Component
Biological speciesEscherichia coli 909945-2 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.9 Å
AuthorsThunnissen, A.M.W.H. / Rozeboom, H.J. / Kunzendorf, A. / Poelarends, G.J.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)724.016.002 Netherlands
CitationJournal: Acs Catalysis / Year: 2021
Title: Unlocking Asymmetric Michael Additions in an Archetypical Class I Aldolase by Directed Evolution.
Authors: Kunzendorf, A. / Xu, G. / van der Velde, J.J.H. / Rozeboom, H.J. / Thunnissen, A.W.H. / Poelarends, G.J.
History
DepositionJul 19, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 27, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Deoxyribose-phosphate aldolase
B: Deoxyribose-phosphate aldolase
C: Deoxyribose-phosphate aldolase
D: Deoxyribose-phosphate aldolase
E: Deoxyribose-phosphate aldolase
F: Deoxyribose-phosphate aldolase
G: Deoxyribose-phosphate aldolase
H: Deoxyribose-phosphate aldolase
I: Deoxyribose-phosphate aldolase
J: Deoxyribose-phosphate aldolase
K: Deoxyribose-phosphate aldolase
L: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)345,88325
Polymers344,20512
Non-polymers1,67813
Water22,9691275
1
A: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
E: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
6
F: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
7
G: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
8
H: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
9
I: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,9083
Polymers28,6841
Non-polymers2242
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
10
J: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
11
K: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
12
L: Deoxyribose-phosphate aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8162
Polymers28,6841
Non-polymers1321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)80.270, 92.788, 106.894
Angle α, β, γ (deg.)88.960, 88.830, 89.080
Int Tables number1
Space group name H-MP1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 4 through 19 or resid 23...
21(chain B and (resid 4 through 19 or resid 23...
31(chain C and (resid 4 through 19 or resid 23...
41(chain D and (resid 4 through 19 or resid 23...
51(chain E and (resid 4 through 19 or resid 23...
61(chain F and (resid 4 through 19 or resid 23...
71(chain G and (resid 4 through 19 or resid 23...
81(chain H and (resid 4 through 39 or resid 41...
91(chain I and (resid 4 through 39 or resid 41...
101(chain J and (resid 4 through 39 or resid 41...
111(chain K and (resid 4 through 19 or resid 23...
121(chain L and (resid 4 through 132 or resid 134 through 206 or resid 208 through 249))

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11LEULEUTHRTHR(chain A and (resid 4 through 19 or resid 23...AA4 - 194 - 19
12ASPASPPROPRO(chain A and (resid 4 through 19 or resid 23...AA23 - 3923 - 39
13GLYGLYALAALA(chain A and (resid 4 through 19 or resid 23...AA41 - 13241 - 132
14VALVALALAALA(chain A and (resid 4 through 19 or resid 23...AA134 - 206134 - 206
15THRTHRGLYGLY(chain A and (resid 4 through 19 or resid 23...AA208 - 249208 - 249
21LEULEUTHRTHR(chain B and (resid 4 through 19 or resid 23...BB4 - 194 - 19
22ASPASPPROPRO(chain B and (resid 4 through 19 or resid 23...BB23 - 3923 - 39
23THRTHR9Y69Y6(chain B and (resid 4 through 19 or resid 23...BB - N2 - 3012
24THRTHR9Y69Y6(chain B and (resid 4 through 19 or resid 23...BB - N2 - 3012
25VALVALALAALA(chain B and (resid 4 through 19 or resid 23...BB134 - 206134 - 206
26THRTHRGLYGLY(chain B and (resid 4 through 19 or resid 23...BB208 - 249208 - 249
31LEULEUTHRTHR(chain C and (resid 4 through 19 or resid 23...CC4 - 194 - 19
32ASPASPPROPRO(chain C and (resid 4 through 19 or resid 23...CC23 - 3923 - 39
33ASPASP9Y69Y6(chain C and (resid 4 through 19 or resid 23...CC - O3 - 3013
34ASPASP9Y69Y6(chain C and (resid 4 through 19 or resid 23...CC - O3 - 3013
35VALVALALAALA(chain C and (resid 4 through 19 or resid 23...CC134 - 206134 - 206
36THRTHRGLYGLY(chain C and (resid 4 through 19 or resid 23...CC208 - 249208 - 249
41LEULEUTHRTHR(chain D and (resid 4 through 19 or resid 23...DD4 - 194 - 19
42ASPASPPROPRO(chain D and (resid 4 through 19 or resid 23...DD23 - 3923 - 39
43ASPASP9Y69Y6(chain D and (resid 4 through 19 or resid 23...DD - P3 - 3013
44ASPASP9Y69Y6(chain D and (resid 4 through 19 or resid 23...DD - P3 - 3013
45VALVALALAALA(chain D and (resid 4 through 19 or resid 23...DD134 - 206134 - 206
46THRTHRGLYGLY(chain D and (resid 4 through 19 or resid 23...DD208 - 249208 - 249
51LEULEUTHRTHR(chain E and (resid 4 through 19 or resid 23...EE4 - 194 - 19
52ASPASPPROPRO(chain E and (resid 4 through 19 or resid 23...EE23 - 3923 - 39
53LEULEU9Y69Y6(chain E and (resid 4 through 19 or resid 23...EE - Q4 - 3014
54LEULEU9Y69Y6(chain E and (resid 4 through 19 or resid 23...EE - Q4 - 3014
55VALVALALAALA(chain E and (resid 4 through 19 or resid 23...EE134 - 206134 - 206
56THRTHRGLYGLY(chain E and (resid 4 through 19 or resid 23...EE208 - 249208 - 249
61LEULEUTHRTHR(chain F and (resid 4 through 19 or resid 23...FF4 - 194 - 19
62ASPASPPROPRO(chain F and (resid 4 through 19 or resid 23...FF23 - 3923 - 39
63ASPASP9Y69Y6(chain F and (resid 4 through 19 or resid 23...FF - R3 - 3013
64ASPASP9Y69Y6(chain F and (resid 4 through 19 or resid 23...FF - R3 - 3013
65VALVALALAALA(chain F and (resid 4 through 19 or resid 23...FF134 - 206134 - 206
66THRTHRGLYGLY(chain F and (resid 4 through 19 or resid 23...FF208 - 249208 - 249
71LEULEUTHRTHR(chain G and (resid 4 through 19 or resid 23...GG4 - 194 - 19
72ASPASPPROPRO(chain G and (resid 4 through 19 or resid 23...GG23 - 3923 - 39
73ASPASP9Y69Y6(chain G and (resid 4 through 19 or resid 23...GG - S3 - 3013
74ASPASP9Y69Y6(chain G and (resid 4 through 19 or resid 23...GG - S3 - 3013
75VALVALALAALA(chain G and (resid 4 through 19 or resid 23...GG134 - 206134 - 206
76THRTHRGLYGLY(chain G and (resid 4 through 19 or resid 23...GG208 - 249208 - 249
81LEULEUPROPRO(chain H and (resid 4 through 39 or resid 41...HH4 - 394 - 39
82GLYGLYALAALA(chain H and (resid 4 through 39 or resid 41...HH41 - 13241 - 132
83VALVALALAALA(chain H and (resid 4 through 39 or resid 41...HH134 - 206134 - 206
84THRTHRGLYGLY(chain H and (resid 4 through 39 or resid 41...HH208 - 249208 - 249
91LEULEUPROPRO(chain I and (resid 4 through 39 or resid 41...II4 - 394 - 39
92GLYGLYALAALA(chain I and (resid 4 through 39 or resid 41...II41 - 13241 - 132
93VALVALALAALA(chain I and (resid 4 through 39 or resid 41...II134 - 206134 - 206
94THRTHRGLYGLY(chain I and (resid 4 through 39 or resid 41...II208 - 249208 - 249
101LEULEUPROPRO(chain J and (resid 4 through 39 or resid 41...JJ4 - 394 - 39
102GLYGLYALAALA(chain J and (resid 4 through 39 or resid 41...JJ41 - 13241 - 132
103VALVALALAALA(chain J and (resid 4 through 39 or resid 41...JJ134 - 206134 - 206
104THRTHRGLYGLY(chain J and (resid 4 through 39 or resid 41...JJ208 - 249208 - 249
111LEULEUTHRTHR(chain K and (resid 4 through 19 or resid 23...KK4 - 194 - 19
112ASPASPPROPRO(chain K and (resid 4 through 19 or resid 23...KK23 - 3923 - 39
113GLYGLYALAALA(chain K and (resid 4 through 19 or resid 23...KK41 - 13241 - 132
114VALVALALAALA(chain K and (resid 4 through 19 or resid 23...KK134 - 206134 - 206
115THRTHRGLYGLY(chain K and (resid 4 through 19 or resid 23...KK208 - 249208 - 249
121LEULEUALAALA(chain L and (resid 4 through 132 or resid 134 through 206 or resid 208 through 249))LL4 - 1324 - 132
122VALVALALAALA(chain L and (resid 4 through 132 or resid 134 through 206 or resid 208 through 249))LL134 - 206134 - 206
123THRTHRGLYGLY(chain L and (resid 4 through 132 or resid 134 through 206 or resid 208 through 249))LL208 - 249208 - 249

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Components

#1: Protein
Deoxyribose-phosphate aldolase / / DERA / 2-deoxy-D-ribose 5-phosphate aldolase / Phosphodeoxyriboaldolase / Deoxyriboaldolase


Mass: 28683.738 Da / Num. of mol.: 12
Mutation: T18S D22G D24Y C47S F52S T142S K172L T197S P202V A203T V206A S239G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli 909945-2 (bacteria) / Gene: deoC, HMPREF1620_02713 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: V0AAC4, deoxyribose-phosphate aldolase
#2: Chemical
ChemComp-9Y6 / (2E)-3-phenylprop-2-enal / Trans-Cinnamaldehyde / Cinnamaldehyde


Mass: 132.159 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C9H8O / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C3H8O3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1275 / 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: 2.31 Å3/Da / Density % sol: 46.8 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: Protein was concentrated to 16 mg/ml in 10 mM potassium phosphate. Crystals grew from hanging-drop vapour diffusion experiments using 13% PEG 3350, 10% isopropanol and 0.1 M HEPES pH 7.5 as reservoir solution

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.9655 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 6, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9655 Å / Relative weight: 1
ReflectionResolution: 1.9→80.24 Å / Num. obs: 229190 / % possible obs: 94.3 % / Redundancy: 1.6 % / Biso Wilson estimate: 15.21 Å2 / CC1/2: 0.98 / Rmerge(I) obs: 0.112 / Rpim(I) all: 0.112 / Rrim(I) all: 0.158 / Net I/σ(I): 2.5 / Num. measured all: 374113
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
1.9-1.931.70.58718962112960.7190.5870.831.393.8
10.41-80.241.80.042250013850.9950.0420.064.693.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
XDS20210205data reduction
Aimless0.7.7data scaling
PHASER2.8.3phasing
Coot0.9.5model building
PHENIX1.19.2refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7P75
Resolution: 1.9→80.24 Å / SU ML: 0.26 / Cross valid method: THROUGHOUT / σ(F): 1.97 / Phase error: 39.82 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2829 11234 4.94 %
Rwork0.2354 216067 -
obs0.2378 227301 93.56 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 69.01 Å2 / Biso mean: 19.8158 Å2 / Biso min: 6.71 Å2
Refinement stepCycle: final / Resolution: 1.9→80.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms22204 0 114 1275 23593
Biso mean--23.46 28.16 -
Num. residues----2964
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A13531X-RAY DIFFRACTION6.279TORSIONAL
12B13531X-RAY DIFFRACTION6.279TORSIONAL
13C13531X-RAY DIFFRACTION6.279TORSIONAL
14D13531X-RAY DIFFRACTION6.279TORSIONAL
15E13531X-RAY DIFFRACTION6.279TORSIONAL
16F13531X-RAY DIFFRACTION6.279TORSIONAL
17G13531X-RAY DIFFRACTION6.279TORSIONAL
18H13531X-RAY DIFFRACTION6.279TORSIONAL
19I13531X-RAY DIFFRACTION6.279TORSIONAL
110J13531X-RAY DIFFRACTION6.279TORSIONAL
111K13531X-RAY DIFFRACTION6.279TORSIONAL
112L13531X-RAY DIFFRACTION6.279TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.9-1.920.4083340.32217207754193
1.92-1.940.36893930.30987129752293
1.94-1.970.36773530.30677147750092
1.97-1.990.35173180.2897192751093
1.99-2.020.34643590.27997181754093
2.02-2.050.31543740.2817058743292
2.05-2.080.33593480.27537125747392
2.08-2.110.33133490.26727149749893
2.11-2.140.3023490.25697152750192
2.14-2.170.32733890.25637056744592
2.17-2.210.35683410.26287159750092
2.21-2.250.30473190.25176983730291
2.25-2.30.31533630.25217023738691
2.3-2.340.29773300.24117127745792
2.34-2.390.29823730.23477189756293
2.39-2.450.29574020.22727313771595
2.45-2.510.29114180.23647334775296
2.51-2.580.28394150.23057326774196
2.58-2.650.27673960.22837332772896
2.65-2.740.28853430.22437338768195
2.74-2.840.2673600.22947283764395
2.84-2.950.27113690.22147381775096
2.95-3.090.28584230.23787248767195
3.09-3.250.27344170.23357188760594
3.25-3.450.24614060.2177258766495
3.45-3.720.25054110.2087329774096
3.72-4.090.25434080.20997215762394
4.09-4.690.21843760.19147133750993
4.69-5.90.23294200.20827449786997
5.9-80.240.24833780.21347063744192

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