- EMDB-24863: CryoEM map of modular PKS holo-Lsd14 bound to antibody fragment 1... -
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Basic information
Entry
Database: EMDB / ID: EMD-24863
Title
CryoEM map of modular PKS holo-Lsd14 bound to antibody fragment 1B2, stalled at the condensation step, focused refinement of KS-KS'-ACP domains
Map data
Full map
Sample
Complex: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM136258
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM138990
United States
Citation
Journal: Science / Year: 2021 Title: Modular polyketide synthase contains two reaction chambers that operate asynchronously. Authors: Saket R Bagde / Irimpan I Mathews / J Christopher Fromme / Chu-Young Kim / Abstract: Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto ...Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo–electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.
Name: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate
Components
Complex: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate
Name: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)
Organism: Streptomyces lasalocidi (bacteria)
Recombinant expression
Organism: Escherichia coli (E. coli)
Molecular weight
Theoretical: 450 KDa
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
4.2 mg/mL
Buffer
pH: 7.2 Component:
Concentration
Name
100.0 mM
sodium chloride
100.0 mM
citrate
10.0 mM
HEPES
1.0 mM
2-Acetaminoethyl-thio-3-oxobutanoate
0.4 mM
methylmalonyl-CoA
0.8 mM
NADP
0.8 mM
TCEP
Grid
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: Blot for 5 seconds before plunging in liquid ethane..
Details
Size exclusion chromatography purified monodisperse holo-Lsd14-DD* + 1B2 complex was first incubated with KS substrate analog (2-Acetaminoethyl-thio-3-oxobutanoate) and TCEP at 30C for 20 minutes, followed by addition of NADP and methylmalonyl-CoA. The sample was further incubated at 30C for 15 minutes before applying to cryoEM grids.
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 1278 / Average exposure time: 2.2 sec. / Average electron dose: 53.0 e/Å2 / Details: Images were collected as 50 frame movies.
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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