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- EMDB-35985: Cryo-EM structure of starch degradation complex of BAM1-LSF1-MDH -

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Basic information

Entry
Database: EMDB / ID: EMD-35985
TitleCryo-EM structure of starch degradation complex of BAM1-LSF1-MDH
Map data
Sample
  • Complex: BAM1-LSF1-MDH complex
    • Protein or peptide: Beta-amylase 1, chloroplastic
    • Protein or peptide: Malate dehydrogenase, chloroplastic
    • Protein or peptide: Phosphoglucan phosphatase LSF1, chloroplastic
Keywordsamylase / starch degradation / HYDROLASE
Function / homology
Function and homology information


chloroplast starch grain / chloroplast protein-transporting ATPase activity / starch grain / Ycf2/FtsHi complex / plastid stroma / carbohydrate phosphatase activity / protein import into chloroplast stroma / : / chloroplast inner membrane / stromule ...chloroplast starch grain / chloroplast protein-transporting ATPase activity / starch grain / Ycf2/FtsHi complex / plastid stroma / carbohydrate phosphatase activity / protein import into chloroplast stroma / : / chloroplast inner membrane / stromule / chloroplast organization / beta-amylase / beta-amylase activity / amylopectin maltohydrolase activity / starch catabolic process / protein tyrosine/serine/threonine phosphatase activity / malate dehydrogenase / embryo development ending in seed dormancy / L-malate dehydrogenase (NAD+) activity / response to water deprivation / malate metabolic process / Hydrolases; Acting on ester bonds; Phosphoric-monoester hydrolases / plant-type vacuole / apoplast / chloroplast envelope / plastid / chloroplast stroma / dephosphorylation / tricarboxylic acid cycle / response to cold / chloroplast / defense response to bacterium / mitochondrion / cytosol
Similarity search - Function
Laforin-like, dual specificity phosphatase domain / Glycoside hydrolase, family 14B, plant / Malate dehydrogenase, type 1 / Malate dehydrogenase, active site / Malate dehydrogenase active site signature. / Beta-amylase active site 2. / Glycoside hydrolase, family 14, conserved site / Beta-amylase active site 1. / Glycoside hydrolase, family 14 / Glycosyl hydrolase family 14 ...Laforin-like, dual specificity phosphatase domain / Glycoside hydrolase, family 14B, plant / Malate dehydrogenase, type 1 / Malate dehydrogenase, active site / Malate dehydrogenase active site signature. / Beta-amylase active site 2. / Glycoside hydrolase, family 14, conserved site / Beta-amylase active site 1. / Glycoside hydrolase, family 14 / Glycosyl hydrolase family 14 / AMP-activated protein kinase, glycogen-binding domain / Glycogen recognition site of AMP-activated protein kinase / Dual specificity phosphatase, catalytic domain / Dual specificity phosphatase, catalytic domain / Dual specificity phosphatase, catalytic domain / Dual specificity protein phosphatase domain / Dual specificity protein phosphatase domain profile. / Lactate/malate dehydrogenase, N-terminal / Lactate/malate dehydrogenase, C-terminal / lactate/malate dehydrogenase, NAD binding domain / lactate/malate dehydrogenase, alpha/beta C-terminal domain / Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal / Protein-tyrosine phosphatase-like / PDZ superfamily / Immunoglobulin E-set / Glycoside hydrolase superfamily / NAD(P)-binding domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Phosphoglucan phosphatase LSF1, chloroplastic / Beta-amylase 1, chloroplastic / Malate dehydrogenase, chloroplastic
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsGuan ZY / Liu J / Yan JJ
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Plant Cell / Year: 2023
Title: The LIKE SEX FOUR 1-malate dehydrogenase complex functions as a scaffold to recruit β-amylase to promote starch degradation.
Authors: Jian Liu / Xuecui Wang / Zeyuan Guan / Menglong Wu / Xinyue Wang / Rong Fan / Fei Zhang / Junjun Yan / Yanjun Liu / Delin Zhang / Ping Yin / Junjie Yan /
Abstract: In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to ...In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including β-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit β-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation.
History
DepositionApr 21, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateJan 10, 2024-
Current statusJan 10, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35985.png

Downloads & links

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Map

FileDownload / File: emd_35985.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.33
Minimum - Maximum-2.8986595 - 4.3270636
Average (Standard dev.)-0.0002657182 (±0.08420865)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 256.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35985_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35985_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : BAM1-LSF1-MDH complex

EntireName: BAM1-LSF1-MDH complex
Components
  • Complex: BAM1-LSF1-MDH complex
    • Protein or peptide: Beta-amylase 1, chloroplastic
    • Protein or peptide: Malate dehydrogenase, chloroplastic
    • Protein or peptide: Phosphoglucan phosphatase LSF1, chloroplastic

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Supramolecule #1: BAM1-LSF1-MDH complex

SupramoleculeName: BAM1-LSF1-MDH complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Beta-amylase 1, chloroplastic

MacromoleculeName: Beta-amylase 1, chloroplastic / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: beta-amylase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 56.111992 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH SDEVDAHMGV PVFVMMPLDS VTMGNTVNRR KAMKASLQAL KSAGVEGIMI DVWWGLVEKE SPGTYNWGG YNELLELAKK LGLKVQAVMS FHQCGGNVGD SVTIPLPQWV VEEVDKDPDL AYTDQWGRRN HEYISLGADT L PVLKGRTP ...String:
MGSSHHHHHH SSGLVPRGSH SDEVDAHMGV PVFVMMPLDS VTMGNTVNRR KAMKASLQAL KSAGVEGIMI DVWWGLVEKE SPGTYNWGG YNELLELAKK LGLKVQAVMS FHQCGGNVGD SVTIPLPQWV VEEVDKDPDL AYTDQWGRRN HEYISLGADT L PVLKGRTP VQCYADFMRA FRDNFKHLLG ETIVEIQVGM GPAGELRYPS YPEQEGTWKF PGIGAFQCYD KYSLSSLKAA AE TYGKPEW GSTGPTDAGH YNNWPEDTQF FKKEGGGWNS EYGDFFLSWY SQMLLDHGER ILSSAKSIFE NMGVKISVKI AGI HWHYGT RSHAPELTAG YYNTRFRDGY LPIAQMLARH NAIFNFTCIE MRDHEQPQDA LCAPEKLVNQ VALATLAAEV PLAG ENALP RYDDYAHEQI LKASALNLDQ NNEGEPREMC AFTYLRMNPE LFQADNWGKF VAFVKKMGEG RDSHRCREEV EREAE HFVH VTQPLVQEAA VALTH

UniProtKB: Beta-amylase 1, chloroplastic

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Macromolecule #2: Malate dehydrogenase, chloroplastic

MacromoleculeName: Malate dehydrogenase, chloroplastic / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: malate dehydrogenase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 34.120137 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: ASYKVAVLGA AGGIGQPLSL LIKMSPLVST LHLYDIANVK GVAADLSHCN TPSQVRDFTG PSELADCLKD VNVVVIPAGV PRKPGMTRD DLFNINANIV KTLVEAVAEN CPNAFIHIIS NPVNSTVPIA AEVLKKKGVY DPKKLFGVTT LDVVRANTFV S QKKNLKLI ...String:
ASYKVAVLGA AGGIGQPLSL LIKMSPLVST LHLYDIANVK GVAADLSHCN TPSQVRDFTG PSELADCLKD VNVVVIPAGV PRKPGMTRD DLFNINANIV KTLVEAVAEN CPNAFIHIIS NPVNSTVPIA AEVLKKKGVY DPKKLFGVTT LDVVRANTFV S QKKNLKLI DVDVPVIGGH AGITILPLLS KTKPSVNFTD EEIQELTVRI QNAGTEVVDA KAGAGSATLS MAYAAARFVE SS LRALDGD GDVYECSFVE STLTDLPFFA SRVKIGKNGL EAVIESDLQG LTEYEQKALE ALKVELKASI DKGVAFANKP AAA AAN

UniProtKB: Malate dehydrogenase, chloroplastic

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Macromolecule #3: Phosphoglucan phosphatase LSF1, chloroplastic

MacromoleculeName: Phosphoglucan phosphatase LSF1, chloroplastic / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Phosphoric-monoester hydrolases
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 59.231191 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: KMNLNEYMVT LEKPLGIRFA LSADGKIFVH AIKKGSNAEK ARIIMVGDTL KKASDSSGGT LVEIKDFGDT KKMLVEKTGS FSLVLERPF SPFPIQYLLH LSDLDLLYNR GRVSFVTWNK NLLSSNLRAS SQGSGNSGYA AFSSKFFTPQ GWKLLNRQSN S FQSGTKKN ...String:
KMNLNEYMVT LEKPLGIRFA LSADGKIFVH AIKKGSNAEK ARIIMVGDTL KKASDSSGGT LVEIKDFGDT KKMLVEKTGS FSLVLERPF SPFPIQYLLH LSDLDLLYNR GRVSFVTWNK NLLSSNLRAS SQGSGNSGYA AFSSKFFTPQ GWKLLNRQSN S FQSGTKKN ILSPPISPLV SVFSEDVPGD GEWGYGNFPL EEYIKALDRS KGELSYNHAL GMRYSKITEQ IYVGSCIQTE ED VENLSEA GITAILNFQG GTEAQNWGID SQSINDACQK SEVLMINYPI KDADSFDLRK KLPLCVGLLL RLLKKNHRVF VTC TTGFDR SSACVIAYLH WMTDTSLHAA YSFVTGLHAC KPDRPAIAWA TWDLIAMVDD GKHDGTPTHS VTFVWNGHEG EEVL LVGDF TGNWKEPIKA THKGGPRFET EVRLTQGKYY YKYIINGDWR HSATSPTERD DRGNTNNIIV VGDVANVRPT IQQPR KDAN IIKVIERVLT ESERFRLAKA ARCIAFSVCP IRLCPKSLEH HHHHH

UniProtKB: Phosphoglucan phosphatase LSF1, chloroplastic

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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

Concentration1 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 305907
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD

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