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- PDB-6are: Aspergillus fumigatus Cytosolic Thiolase in complex with two tetr... -

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Basic information

Entry
Database: PDB / ID: 6are
TitleAspergillus fumigatus Cytosolic Thiolase in complex with two tetrahedral reaction intermediates and ammonium ions
Components
  • Acetyl-CoA acetyltransferase
  • Acetyl-CoA acetyltransferase, acetylated enzyme
KeywordsTRANSFERASE / thiolase / Claisen Condensation / reaction intermediate / monovalent cation binding
Function / homology
Function and homology information


acetyl-CoA C-acetyltransferase / ergosterol biosynthetic process / acetyl-CoA C-acetyltransferase activity / mitochondrion / metal ion binding / cytosol
Similarity search - Function
Thiolase, conserved site / Thiolases signature 2. / Thiolase, acyl-enzyme intermediate active site / Thiolases acyl-enzyme intermediate signature. / Thiolase, C-terminal / Thiolase, C-terminal domain / Thiolase / Thiolase, N-terminal / Thiolase, N-terminal domain / Thiolase/Chalcone synthase ...Thiolase, conserved site / Thiolases signature 2. / Thiolase, acyl-enzyme intermediate active site / Thiolases acyl-enzyme intermediate signature. / Thiolase, C-terminal / Thiolase, C-terminal domain / Thiolase / Thiolase, N-terminal / Thiolase, N-terminal domain / Thiolase/Chalcone synthase / Peroxisomal Thiolase; Chain A, domain 1 / Thiolase-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ACETYL COENZYME *A / ACETATE ION / COENZYME A / AMMONIUM ION / Acetyl-CoA acetyltransferase erg10B, cytosolic
Similarity search - Component
Biological speciesAspergillus fumigatus Af293 (mold)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.75 Å
Model detailsCrystallized in the presence of acetyl-CoA, K+, and NH4+. 3 active sites contain Ac-Cys92+CoA: 1 ...Crystallized in the presence of acetyl-CoA, K+, and NH4+. 3 active sites contain Ac-Cys92+CoA: 1 site contains Ac-Cys92+Ac-CoA (alternate conformations).
AuthorsMarshall, A.C. / Bond, C.S. / Bruning, J.B.
CitationJournal: Acs Catalysis / Year: 2018
Title: Structure of Aspergillus fumigatus Cytosolic Thiolase: Trapped Tetrahedral Reaction Intermediates and Activation by Monovalent Cations
Authors: Marshall, A.C. / Bond, C.S. / Bruning, J.B.
History
DepositionAug 22, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 30, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 2, 2025Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_entry_details.has_protein_modification / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Acetyl-CoA acetyltransferase, acetylated enzyme
B: Acetyl-CoA acetyltransferase, acetylated enzyme
C: Acetyl-CoA acetyltransferase
D: Acetyl-CoA acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,66824
Polymers164,1154
Non-polymers3,55220
Water24,5001360
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area21350 Å2
ΔGint-106 kcal/mol
Surface area48420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.360, 106.590, 110.840
Angle α, β, γ (deg.)90.000, 104.270, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 2 types, 4 molecules ABCD

#1: Protein Acetyl-CoA acetyltransferase, acetylated enzyme


Mass: 41049.887 Da / Num. of mol.: 2 / Mutation: C92SCY
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus fumigatus Af293 (mold) / Strain: ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100 / Gene: AFUA_8G04000 / Plasmid: pET-57-DEST / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q4WCL5, acetyl-CoA C-acetyltransferase
#2: Protein Acetyl-CoA acetyltransferase


Mass: 41007.848 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus fumigatus Af293 (mold) / Strain: ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100 / Gene: AFUA_8G04000 / Plasmid: pET-57-DEST / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q4WCL5, acetyl-CoA C-acetyltransferase

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Non-polymers , 6 types, 1380 molecules

#3: Chemical ChemComp-COA / COENZYME A


Mass: 767.534 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C21H36N7O16P3S
#4: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
#5: Chemical
ChemComp-NH4 / AMMONIUM ION


Mass: 18.038 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: H4N
#6: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#7: Chemical ChemComp-ACO / ACETYL COENZYME *A


Mass: 809.571 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C23H38N7O17P3S
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1360 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsThe protein was co-crystallised with acetyl-CoA. It is an acetoacetyl-CoA thiolase, producing ...The protein was co-crystallised with acetyl-CoA. It is an acetoacetyl-CoA thiolase, producing acetoacetyl-CoA from two molecules of acetyl-CoA. The reaction proceeds via two acetyl transfer steps: (1) Ac-CoA -> enzyme, producing CoA and acetylated enzyme (SCY92), and (2) Ac-enzyme -> Ac-CoA, producing acetoacetyl-CoA. The enzyme is a homotetramer, thus 4 active sites per AU. For two of the active sites (chains A and B), the density indicates that CoA and SCY are present (after (1)). For chain D, the tetrahedral intermediate that occurs during the first acetyl transfer is trapped. To model this we used three separate entities: cys92, an acetyl group, and CoA. Covalent bonds were defined between these during refinement. For chain C, the tetrahedral intermediate that occurs during the second acetyl transfer is trapped. To model this we used cys92, an acetyl group, and acetyl-CoA.
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: 2.49 Å3/Da / Density % sol: 50.56 %
Crystal growTemperature: 289 K / Method: vapor diffusion / pH: 6.5 / Details: 25% PEG 3350, 0.2M AmS04, 0.1M Bis-Tris pH 6.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX1 / Wavelength: 0.9537 Å
DetectorType: ADSC QUANTUM 210r / Detector: CCD / Date: Feb 25, 2017
RadiationMonochromator: Silicon Double Crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 1.75→31.54 Å / Num. obs: 160937 / % possible obs: 99.6 % / Redundancy: 7.3 % / Biso Wilson estimate: 13.03 Å2 / CC1/2: 0.968 / Rmerge(I) obs: 0.385 / Rpim(I) all: 0.152 / Rrim(I) all: 0.414 / Net I/σ(I): 4.1 / Num. measured all: 1171578 / Scaling rejects: 1205
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
1.75-1.787.11.9715604179290.3680.7932.126199
9.59-31.546.30.202636110130.9560.0850.2198.797.2

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation6.34 Å107.47 Å
Translation6.34 Å107.47 Å

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Processing

Software
NameVersionClassification
PHENIXrefinement
MOSFLMdata collection
Aimless0.5.17data scaling
PHASER2.5.6phasing
PDB_EXTRACT3.22data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.75→31.288 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 23.89 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2315 8013 4.98 %
Rwork0.1944 152876 -
obs0.1963 160889 99.57 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 75.87 Å2 / Biso mean: 19.7061 Å2 / Biso min: 1.99 Å2
Refinement stepCycle: final / Resolution: 1.75→31.288 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11358 0 215 1362 12935
Biso mean--43.94 25.78 -
Num. residues----1588
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00811871
X-RAY DIFFRACTIONf_angle_d0.98416187
X-RAY DIFFRACTIONf_chiral_restr0.0541905
X-RAY DIFFRACTIONf_plane_restr0.0062113
X-RAY DIFFRACTIONf_dihedral_angle_d14.934296
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 (%)
1.75-1.76990.34792360.34415052528899
1.7699-1.79070.35152830.32925055533899
1.7907-1.81250.34162760.31445044532099
1.8125-1.83550.33492700.30795057532799
1.8355-1.85960.32392830.30025002528599
1.8596-1.88510.32212800.28395053533399
1.8851-1.9120.30012750.27275015529099
1.912-1.94060.3142420.25875162540499
1.9406-1.97090.2632870.2475014530199
1.9709-2.00320.27432760.24175081535799
2.0032-2.03770.27712550.24795077533299
2.0377-2.07480.29412870.24775038532599
2.0748-2.11470.28332650.236450735338100
2.1147-2.15780.25172500.22895089533999
2.1578-2.20470.25632560.215451075363100
2.2047-2.2560.24392670.214950895356100
2.256-2.31240.27242500.203751425392100
2.3124-2.37490.23252580.196251055363100
2.3749-2.44480.26572800.190850615341100
2.4448-2.52370.22422830.185951055388100
2.5237-2.61380.21182560.180351095365100
2.6138-2.71840.23753010.183650705371100
2.7184-2.8420.24542490.183151605409100
2.842-2.99180.21442540.174251345388100
2.9918-3.1790.20832510.164151225373100
3.179-3.42420.18722770.154251575434100
3.4242-3.76830.18332660.141151365402100
3.7683-4.31240.15072570.126851545411100
4.3124-5.42850.16152600.124251915451100
5.4285-31.29260.14142830.138352225505100
Refinement TLS params.

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

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.7818-0.7745-0.08380.8823-0.01893.79050.02360.08710.0416-0.0477-0.03930.0142-0.0708-0.1050.01780.0143-0.00630.00120.0114-0.02540.1493-29.91189.9714108.8251
20.9508-0.0238-0.02520.5946-0.18410.3103-0.0284-0.14030.01910.04420.01940.0628-0.0371-0.02530.00950.05620.01910.020.0339-0.0060.1117-23.681115.5078118.6397
31.85610.6268-0.56632.82920.38152.3715-0.1243-0.1372-0.4388-0.0784-0.0382-0.02520.43290.02780.12290.0379-0.01740.00190.09340.04890.2956-42.49354.8747113.6453
40.8547-0.15220.01810.70390.05450.4721-0.0063-0.00520.18630.00170.01440.0957-0.0765-0.0896-0.00670.06640.0145-0.00050.0373-0.01040.1665-30.867524.703111.2104
51.33260.3027-1.19481.5281-1.92333.0753-0.07030.03290.1453-0.0030.0109-0.14090.0575-0.11060.01420.0201-0.0024-0.01280.0496-0.00920.15691.713415.7547118.0434
60.9901-0.14990.22640.31250.06650.4903-0.0479-0.202-0.05040.0420.034-0.02440.0236-0.02430.00690.04920.01370.0244-0.0390.01730.1489-7.13156.7081119.8201
71.91970.3799-0.01252.9103-0.34491.0798-0.0389-0.361-0.12280.31560.0084-0.21070.02220.10870.04020.08490.0431-0.01520.10830.03260.1227.36090.5911127.7665
80.5151-0.15630.20730.77540.17830.27-0.0150.0885-0.0363-0.08420.0285-0.08460.05260.0926-0.01260.0679-0.00520.02740.0456-0.00450.14172.95545.0128107.3601
91.33840.4016-0.32380.78410.30672.2222-0.0291-0.0050.28840.0537-0.02280.0698-0.1116-0.12910.03290.09580.01650.0140.26910.03280.1023-39.82742.4311167.5369
101.1809-0.0319-0.73410.5649-0.18321.0255-0.07120.1489-0.0375-0.0670.0174-0.00270.10130.00320.05870.1021-0.00860.01360.27760.02110.0893-29.4196-5.5225159.169
111.69370.28910.06420.8931-0.09011.1031-0.04850.3115-0.086-0.09380.0620.09090.124-0.238-0.00940.1003-0.0310.01420.31290.01460.0901-45.9506-12.4966162.2516
121.01090.5944-0.17511.2586-0.67541.5014-0.0956-0.2158-0.11420.1270.0184-0.10350.18010.28870.04710.08130.06990.02030.41590.06910.1035-14.1069-10.2746169.7488
130.888-0.2309-0.05980.826-0.521.0440.02110.00930.14330.0107-0.0498-0.0676-0.05110.17310.0230.1142-0.02210.01910.26620.03520.1193-21.14811.8605163.2974
142.0115-0.087-0.00740.79510.09671.0775-0.0511-0.37680.37820.08470.1042-0.11-0.11840.4623-0.02580.1543-0.0645-0.02430.462-0.03410.1518-7.97824.2193175.5636
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 0 through 43 )A0 - 43
2X-RAY DIFFRACTION2chain 'A' and (resid 44 through 195 )A44 - 195
3X-RAY DIFFRACTION3chain 'A' and (resid 196 through 216 )A196 - 216
4X-RAY DIFFRACTION4chain 'A' and (resid 217 through 398 )A217 - 398
5X-RAY DIFFRACTION5chain 'B' and (resid 0 through 43 )B0 - 43
6X-RAY DIFFRACTION6chain 'B' and (resid 44 through 195 )B44 - 195
7X-RAY DIFFRACTION7chain 'B' and (resid 196 through 256 )B196 - 256
8X-RAY DIFFRACTION8chain 'B' and (resid 257 through 398 )B257 - 398
9X-RAY DIFFRACTION9chain 'C' and (resid 1 through 68 )C1 - 68
10X-RAY DIFFRACTION10chain 'C' and (resid 69 through 175 )C69 - 175
11X-RAY DIFFRACTION11chain 'C' and (resid 176 through 398 )C176 - 398
12X-RAY DIFFRACTION12chain 'D' and (resid 0 through 68 )D0 - 68
13X-RAY DIFFRACTION13chain 'D' and (resid 69 through 175 )D69 - 175
14X-RAY DIFFRACTION14chain 'D' and (resid 176 through 398 )D176 - 398

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