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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-9873 | |||||||||||||||
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Title | Structure of PaaZ, a bifunctional enzyme | |||||||||||||||
![]() | PaaZ is a bifucntional enzyme that performs hydratase and dehydrogenase reactions. This structure is the native protein. This map is final sharpened map used for interpretation. | |||||||||||||||
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Function / homology | ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||||||||
Biological species | ![]() ![]() ![]() | |||||||||||||||
Method | ![]() ![]() | |||||||||||||||
![]() | Gakher L / Vinothkumar KR / Katagihallimath N / Sowdhamini R / Sathyanarayanan N / Cannone G | |||||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() Title: Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway. Authors: Nitish Sathyanarayanan / Giuseppe Cannone / Lokesh Gakhar / Nainesh Katagihallimath / Ramanathan Sowdhamini / Subramanian Ramaswamy / Kutti R Vinothkumar / ![]() ![]() ![]() Abstract: Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been ...Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues. | |||||||||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 250 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.8 KB 20.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 14.3 KB | Display | ![]() |
Images | ![]() | 32.7 KB | ||
Filedesc metadata | ![]() | 7 KB | ||
Others | ![]() ![]() | 212.6 MB 212.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6jqlMC ![]() 9874C ![]() 9875C ![]() 9876C ![]() 6jqmC ![]() 6jqnC ![]() 6jqoC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | PaaZ is a bifucntional enzyme that performs hydratase and dehydrogenase reactions. This structure is the native protein. This map is final sharpened map used for interpretation. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: This map is one of the half-maps after refinement.
File | emd_9873_additional_1.map | ||||||||||||
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Annotation | This map is one of the half-maps after refinement. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: This map is one of the half-maps after refinement.
File | emd_9873_additional_2.map | ||||||||||||
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Annotation | This map is one of the half-maps after refinement. | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : PaaZ
Entire | Name: PaaZ |
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Components |
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-Supramolecule #1: PaaZ
Supramolecule | Name: PaaZ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: PaaZ is a bifunctional enzyme that has hydrolase and dehydrogenase activity. |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 440 KDa |
-Macromolecule #1: Bifunctional protein PaaZ
Macromolecule | Name: Bifunctional protein PaaZ / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: ![]() |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 73.969391 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MGHHHHHHQQ LASFLSGTWQ SGRGRSRLIH HAISGEALWE VTSEGLDMAA ARQFAIEKGA PALRAMTFIE RAAMLKAVAK HLLSEKERF YALSAQTGAT RADSWVDIEG GIGTLFTYAS LGSRELPDDT LWPEDELIPL SKEGGFAARH LLTSKSGVAV H INAFNFPC ...String: MGHHHHHHQQ LASFLSGTWQ SGRGRSRLIH HAISGEALWE VTSEGLDMAA ARQFAIEKGA PALRAMTFIE RAAMLKAVAK HLLSEKERF YALSAQTGAT RADSWVDIEG GIGTLFTYAS LGSRELPDDT LWPEDELIPL SKEGGFAARH LLTSKSGVAV H INAFNFPC WGMLEKLAPT WLGGMPAIIK PATATAQLTQ AMVKSIVDSG LVPEGAISLI CGSAGDLLDH LDSQDVVTFT GS AATGQML RVQPNIVAKS IPFTMEADSL NCCVLGEDVT PDQPEFALFI REVVREMTTK AGQKCTAIRR IIVPQALVNA VSD ALVARL QKVVVGDPAQ EGVKMGALVN AEQRADVQEK VNILLAAGCE IRLGGQADLS AAGAFFPPTL LYCPQPDETP AVHA TEAFG PVATLMPAQN QRHALQLACA GGGSLAGTLV TADPQIARQF IADAARTHGR IQILNEESAK ESTGHGSPLP QLVHG GPGR AGGGEELGGL RAVKHYMQRT AVQGSPTMLA AISKQWVRGA KVEEDRIHPF RKYFEELQPG DSLLTPRRTM TEADIV NFA CLSGDHFYAH MDKIAAAESI FGERVVHGYF VLSAAAGLFV DAGVGPVIAN YGLESLRFIE PVKPGDTIQV RLTCKRK TL KKQRSAEEKP TGVVEWAVEV FNQHQTPVAL YSILTLVARQ HGDFVD UniProtKB: ![]() |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Concentration | 0.015 mg/mL | ||||||
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Buffer | pH: 7.4 Component:
Details: Protein was purified and kept in 25mM Hepes buffer and 50 mM NaCl | ||||||
Grid | Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa Details: The Ultrafoil grids was glow discharged in air for 5 minutes. Subsequently, 0.2 mg/ml of graphene oxide solution was placed and washed twice with water. The grids were air dried and used directly for freezing. | ||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV Details: blotting force 10, blotting time 4 sec, waiting time 15 sec, drying time 0, blotting times 1.. | ||||||
Details | The peak fraction from gel filtration was used for grid preparation. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated magnification: 132075 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Temperature | Min: 80.0 K / Max: 80.0 K |
Details | Data was collected with EPU software |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 552 / Average exposure time: 60.0 sec. / Average electron dose: 27.0 e/Å2 Details: The 60 second exposure was saved into 75 frames with each frame ~0.36 e-. The frames were then grouped into 3 for alignment and summed images were used for data processing |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Details | Real space refinement with secondary structure enabled, minimization and adp |
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Refinement | Space: REAL / Protocol: OTHER / Overall B value: 44.4 |
Output model | ![]() PDB-6jql: |