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- EMDB-71497: Antibody (1B2) Bound Crosslinked Rifamycin Synthetase Module 1 wi... -
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Open data
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Basic information
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Title | Antibody (1B2) Bound Crosslinked Rifamycin Synthetase Module 1 with a C-terminal Type II Thioesterase | |||||||||
![]() | Antibody (1B2) Bound Crosslinked Rifamycin Synthetase Module 1 with a C-terminal Type II Thioesterase | |||||||||
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![]() | Polyketide Synthase Module / Antibody (Fab) / Transferase-Immune System complex | |||||||||
Function / homology | ![]() 6-deoxyerythronolide-B synthase / fatty acid synthase activity / lipid biosynthetic process / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / antibiotic biosynthetic process / fatty acid biosynthetic process / hydrolase activity Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.96 Å | |||||||||
![]() | Cogan DP / Liu C / West RC / Chen M | |||||||||
Funding support | 1 items
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![]() | ![]() Title: Molecular Basis for Asynchronous Chain Elongation During Rifamycin Antibiotic Biosynthesis. Authors: Chengli Liu / Ryan C West / Muyuan Chen / Whitaker Cohn / George Wang / Aryan M Mandot / Selena Kim / Dillon P Cogan / ![]() Abstract: The rifamycin synthetase (RIFS) from the bacterium is a large (3.5 MDa) multienzyme system that catalyzes over 40 chemical reactions to generate a complex precursor to the antibiotic rifamycin B. It ...The rifamycin synthetase (RIFS) from the bacterium is a large (3.5 MDa) multienzyme system that catalyzes over 40 chemical reactions to generate a complex precursor to the antibiotic rifamycin B. It is considered a hybrid enzymatic assembly line and consists of an N-terminal nonribosomal peptide synthetase loading module followed by a decamodular polyketide synthase (PKS). While the biosynthetic functions are known for each enzymatic domain of RIFS, structural and biochemical analyses of this system from purified components are relatively scarce. Here, we examine the biosynthetic mechanism of RIFS through complementary crosslinking, kinetic, and structural analyses of its first PKS module (M1). Thiol-selective crosslinking of M1 provided a plausible molecular basis for previously observed conformational asymmetry with respect to ketosynthase (KS)-substrate carrier protein (CP) interactions during polyketide chain elongation. Our data suggest that C-terminal dimeric interfaces-which are ubiquitous in bacterial PKSs-force their adjacent CP domains to co-migrate between two equivalent KS active site chambers. Cryogenic electron microscopy analysis of M1 further supported this observation while uncovering its unique architecture. Single-turnover kinetic analysis of M1 indicated that although removal of C-terminal dimeric interfaces supported 2-fold greater KS-CP interactions, it did not increase the partial product occupancy of the homodimeric protein. Our findings cast light on molecular details of natural antibiotic biosynthesis that will aid in the design of artificial megasynth(et)ases with untold product structures and bioactivities. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 407.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 24.5 KB 24.5 KB | Display Display | ![]() |
Images | ![]() | 39.1 KB | ||
Filedesc metadata | ![]() | 8.1 KB | ||
Others | ![]() ![]() ![]() | 18.5 MB 765 MB 765 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1.2 MB | Display | ![]() |
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Full document | ![]() | 1.2 MB | Display | |
Data in XML | ![]() | 21 KB | Display | |
Data in CIF | ![]() | 25.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9pc6MC ![]() 9patC ![]() 9pavC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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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 | Antibody (1B2) Bound Crosslinked Rifamycin Synthetase Module 1 with a C-terminal Type II Thioesterase | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.73 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Additional Map
File | emd_71497_additional_1.map | ||||||||||||
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Annotation | Additional Map | ||||||||||||
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Density Histograms |
-Half map: Half Map A
File | emd_71497_half_map_1.map | ||||||||||||
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Annotation | Half Map A | ||||||||||||
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Density Histograms |
-Half map: Half Map B
File | emd_71497_half_map_2.map | ||||||||||||
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Annotation | Half Map B | ||||||||||||
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Density Histograms |
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Sample components
-Entire : Two antibody fragments (Fabs) in complex with homodimeric, crossl...
Entire | Name: Two antibody fragments (Fabs) in complex with homodimeric, crosslinked polyketide synthase module 1 of the rifamycin synthetase fused with a C-terminal type II thioesterase |
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Components |
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-Supramolecule #1: Two antibody fragments (Fabs) in complex with homodimeric, crossl...
Supramolecule | Name: Two antibody fragments (Fabs) in complex with homodimeric, crosslinked polyketide synthase module 1 of the rifamycin synthetase fused with a C-terminal type II thioesterase type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 495.04131 KDa |
-Macromolecule #1: 6-deoxyerythronolide-B synthase,RifR
Macromolecule | Name: 6-deoxyerythronolide-B synthase,RifR / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: 6-deoxyerythronolide-B synthase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 196.008141 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MASTDSEKVA EYLRRATLDL RAARQRIREL EGEPIAIVGM ACRLPGGVAS PEDLWRLVAE RVDAVSEFPG DRGWDLDSLI DPDRERAGT SYVGQGGFLH DAGEFDAGFF GISPREAVAM DPQQRLLLET SWEALENAGV DPIALKGTDT GVFSGLMGQG Y GSGAVAPE ...String: MASTDSEKVA EYLRRATLDL RAARQRIREL EGEPIAIVGM ACRLPGGVAS PEDLWRLVAE RVDAVSEFPG DRGWDLDSLI DPDRERAGT SYVGQGGFLH DAGEFDAGFF GISPREAVAM DPQQRLLLET SWEALENAGV DPIALKGTDT GVFSGLMGQG Y GSGAVAPE LEGFVTTGVA SSVASGRVSY VLGLEGPAVT VDTACSSSLV AMHLAAQALR QGECSMALAG GVTVMATPGS FV EFSRQRA LAPDGRCKAF AAAADGTGWS EGVGVVVLER LSVARERGHR ILAVLRGSAV NQDGASNGLT APNGLSQQRV IRR ALAAAG LAPSDVDVVE AHGTGTTLGD PIEAQALLAT YGQERKQPLW LGSLKSNIGH AQAAAGVAGV IKMVQALRHE TLPP TLHVD KPTLEVDWSA GAIELLTEAR AWPRNGRPRR AGVSSFGVSG TNAHLILEEA PAEEPVAAPE LPVVPLVVSA RSTES LSGQ AERLASLLEG DVSLTEVAGA LVSRRAVLDE RAVVVAGSRE EAVTGLRALN TAGSGTPGKV VWVFPGQGTQ WAGMGR ELL AESPVFAERI AECAAALAPW IDWSLVDVLR GEGDLGRVDV LQPACFAVMV GLAAVWESVG VRPDAVVGHS QGEIAAA CV SGALSLEDAA KVVALRSQAI AAELSGRGGM ASVALGEDDV VSRLVDGVEV AAVNGPSSVV IAGDAHALDA TLEILSGE G IRVRRVAVDY ASHTRHVEDI RDTLAETLAG ISAQAPAVPF YSTVTSEWVR DAGVLDGGYW YRNLRNQVRF GAAATALLE QGHTVFVEVS AHPVTVQPLS ELTGDAIGTL RREDGGLRRL LASMGELFVR GIDVDWTAMV PAAGWVDLPT YAFEHRHYWL EPAEPASAG DPLLGTVVST PGSDRLTAVA QWSRRAQPWA VDGLVPNAAL VEAAIRLGDL AGTPVVGELV VDAPVVLPRR G SREVQLIV GEPGEQRRRP IEVFSREADE PWTRHAHGTL APAAAAVPEP AAAGDATDVT VAGLRDADRY GIHPALLDAA VR TVVGDDL LPSVWTGVSL LASGATAVTV TPTATGLRLT DPAGQPVLTV ESVRGTPFVA EQGTTDALFR VDWPEIPLPT AET ADFLPY EATSAEATLS ALQAWLADPA ETRLAVVTGD CTEPGAAAIW GLVRSAQSEH PGRIVLADLD DPAVLPAVVA SGEP QVRVR NGVASVPRLT RVTPRQDARP LDPEGTVLIT GGTGTLGALT ARHLVTAHGV RHLVLVSRRG EAPELQEELT ALGAS VAIA ACDVADRAQL EAVLRAIPAE HPLTAVIHTA GVLDDGVVTE LTPDRLATVR RPKVDAARLL DELTREADLA AFVLFS SAA GVLGNPGQAG YAAANAELDA LARQRNSLDL PAVSIAWGYW ATVSGMTEHL GDADLRRNQR IGMSGLPADE GMALLDA AI ATGGTLVAAK FDVAALRATA KAGGPVPPLL RGLAPLPRRA AAKTASLTER LAGLAETEQA AALLDLVRRH AAEVLGHS G AESVHSGRTF KDAGFD(4HH)LTA VELRNRLAAA TGLTLSPAMI FDYPKPPALA DHLRAKLFGS GGGGSGGGGS HRPEA EKWL RRFERAPDAR ARLVCLPHAG GSASFFFPLA KALAPAVEVL AVQYPGRQDR RHEPPVDSIG GLTNRLLEVL RPFGDR PLA LFGHSMGAII GYELALRMPE AGLPAPVHLF ASGRRAPSRY RDDDVRGASD ERLVAELRKL GGSDAAMLAD PELLAMV LP AIRSDYRAVE TYRHEPGRRV DCPVTVFTGD HDPRVSVGEA RAWEEHTTGP ADLRVLPGGH FFLVDQAAPM IATMTEKL A GPALTGSTGG NSGNSSSVDK LAAALEHHHH HH UniProtKB: 6-deoxyerythronolide-B synthase, RifR |
-Macromolecule #2: Antibody Fragment 1B2 Heavy Chain
Macromolecule | Name: Antibody Fragment 1B2 Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 26.447611 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MAEVQLVQSG GGLVQPGRSL RLSCTASGFT FGDYAMSWVR QAPGKGLEWV GFIRSKAYGG TTEYAASVKG RFTISRDDSK SIAYLQMNS LKTEDTAVYY CTRGGTLFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String: MAEVQLVQSG GGLVQPGRSL RLSCTASGFT FGDYAMSWVR QAPGKGLEWV GFIRSKAYGG TTEYAASVKG RFTISRDDSK SIAYLQMNS LKTEDTAVYY CTRGGTLFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCAALVP RGSAHHHHHH AA DYKDDDD KA |
-Macromolecule #3: Antibody Fragment 1B2 Light Chain
Macromolecule | Name: Antibody Fragment 1B2 Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 25.715832 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: LFAIPLVVPF YSHSALDVVM TQSPLSLPVT PGEPASISCR SSQSLLHSNG YNYLDWYLQK PGQSPQLLIY LGSNRASGVP DRFSGSGSG TDFTLKISRV EAEDVGVYYC MQSLQTPRLT FGPGTKVDIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN N FYPRGAKV ...String: LFAIPLVVPF YSHSALDVVM TQSPLSLPVT PGEPASISCR SSQSLLHSNG YNYLDWYLQK PGQSPQLLIY LGSNRASGVP DRFSGSGSG TDFTLKISRV EAEDVGVYYC MQSLQTPRLT FGPGTKVDIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN N FYPRGAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 10 mg/mL | |||||||||
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Buffer | pH: 7.2 Component:
Details: 100 mM citric acid, 10 mM HEPES, pH 7.2 (NaOH) | |||||||||
Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.036000000000000004 kPa | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | TFS GLACIOS |
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Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 7631 / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.677 µm / Nominal defocus min: 0.1 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
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Image processing
-Atomic model buiding 1
Initial model | Chain - Source name: AlphaFold / Chain - Initial model type: in silico model |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | ![]() PDB-9pc6: |