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- EMDB-24873: CryoEM map of modular PKS holo-Lsd14 bound to antibody fragment 1... -

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Basic information

Entry
Database: EMDB / ID: EMD-24873
TitleCryoEM map of modular PKS holo-Lsd14 bound to antibody fragment 1B2, focused refinement of KR domain
Map dataFull 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.
Function / homology
Function and homology information


6-deoxyerythronolide-B synthase / erythronolide synthase activity / macrolide biosynthetic process / DIM/DIP cell wall layer assembly / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / oxidoreductase activity / plasma membrane / cytoplasm
Similarity search - Function
Erythronolide synthase, docking / Erythronolide synthase, docking domain superfamily / Erythronolide synthase, docking / Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / Polyketide synthase, docking domain superfmaily / Polyketide synthase extender module SpnB, Rossmann fold domain / Polyketide synthase, docking domain / Erythronolide synthase docking domain / Zinc-binding dehydrogenase ...Erythronolide synthase, docking / Erythronolide synthase, docking domain superfamily / Erythronolide synthase, docking / Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / Polyketide synthase, docking domain superfmaily / Polyketide synthase extender module SpnB, Rossmann fold domain / Polyketide synthase, docking domain / Erythronolide synthase docking domain / Zinc-binding dehydrogenase / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / : / Polyketide synthase dehydratase domain / Polyketide synthase, dehydratase domain superfamily / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / PKS_PP_betabranch / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Polyketide synthase, ketoreductase domain / KR domain / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase domain superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / Polyketide synthase, enoylreductase domain / Enoylreductase / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase GroES-like domain / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / PKS_KR / Ketosynthase family 3 (KS3) domain profile. / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / GroES-like superfamily / Thiolase-like / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Putative polyketide synthase / 6-deoxyerythronolide-B synthase EryA2, modules 3 and 4
Similarity search - Component
Biological speciesStreptomyces lasalocidi (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsBagde SR / Kim C-Y / Fromme JC
Funding support United States, 3 items
OrganizationGrant numberCountry
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
National Science Foundation (NSF, United States)DMR-1719875 United States
CitationJournal: 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.
History
DepositionSep 13, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24873.png

Downloads & links

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Map

FileDownload / File: emd_24873.map.gz / Format: CCP4 / Size: 184 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 364 pix.
= 390.208 Å		1.07 Å/pix.
x 364 pix.
= 390.208 Å		1.07 Å/pix.
x 364 pix.
= 390.208 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.072 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.025202576 - 0.07110254
Average (Standard dev.)8.830449e-06 (±0.0014947813)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions364364364
Spacing364364364
CellA=B=C: 390.208 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0721.0721.072
M x/y/z364364364
origin x/y/z0.0000.0000.000
length x/y/z390.208390.208390.208
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS364364364
D min/max/mean-0.0250.0710.000

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Supplemental data

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Mask #1

Fileemd_24873_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocessed masked map

Fileemd_24873_additional_1.map
AnnotationPostprocessed masked map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_24873_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_24873_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : Dimeric holo-Lsd14 containing docking domains from DEBS module 3 ...

EntireName: 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.
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.

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Supramolecule #1: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 ...

SupramoleculeName: 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.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Streptomyces lasalocidi (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 450 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.2 mg/mL
BufferpH: 7.2
Component:
ConcentrationName
100.0 mMsodium chloride
100.0 mMcitrate
10.0 mMHEPES
0.4 mMmethylmalonyl-CoA
0.8 mMNADP
0.8 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot for 5 seconds before plunging in liquid ethane..
DetailsSize exclusion chromatography purified monodisperse holo-Lsd14-DD* + 1B2 complex was mixed with native AT substrate methylmalonyl-CoA, NADP and TCEP. This sample was incubated at 30C for 15 minutes before applying to cryo-EM grids.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 2787 / Average exposure time: 3.5 sec. / Average electron dose: 54.0 e/Å2 / Details: Images were collected as 50 frame movies.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 63000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4540000
CTF correctionSoftware:
Namedetails
cryoSPARC (ver. 3.2.0)Patch CTF estimation (multi)
RELION (ver. 3.1)
Startup modelType of model: INSILICO MODEL
In silico model: Used Ab-initio Reconstruction job in cryoSPARC to generate starting map.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 62591
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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