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- EMDB-19742: Full-length human cystathionine beta-synthase with C-terminal 6xH... -

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Basic information

Entry
Database: EMDB / ID: EMD-19742
TitleFull-length human cystathionine beta-synthase with C-terminal 6xHis-tag, SAM bound, activated state, local single particle reconstruction
Map data
Sample
  • Complex: Helical assembly of full-length human cystathionine beta-synthase bound to SAM in the activated state
    • Protein or peptide: cystathionine beta-synthaseCystathionine beta synthase
KeywordsFilament / Allostery / TRANSFERASE
Function / homology
Function and homology information


Cysteine formation from homocysteine / homocysteine catabolic process / modified amino acid binding / cystathionine beta-synthase / cystathionine beta-synthase activity / cysteine biosynthetic process via cystathionine / Metabolism of ingested SeMet, Sec, MeSec into H2Se / homocysteine metabolic process / L-serine catabolic process / carbon monoxide binding ...Cysteine formation from homocysteine / homocysteine catabolic process / modified amino acid binding / cystathionine beta-synthase / cystathionine beta-synthase activity / cysteine biosynthetic process via cystathionine / Metabolism of ingested SeMet, Sec, MeSec into H2Se / homocysteine metabolic process / L-serine catabolic process / carbon monoxide binding / hydrogen sulfide biosynthetic process / cartilage development involved in endochondral bone morphogenesis / L-serine metabolic process / regulation of nitric oxide mediated signal transduction / L-cysteine catabolic process / cysteine biosynthetic process / cerebellum morphogenesis / nitric oxide binding / cysteine biosynthetic process from serine / DNA protection / endochondral ossification / transsulfuration / S-adenosyl-L-methionine binding / response to folic acid / nitrite reductase (NO-forming) activity / superoxide metabolic process / maternal process involved in female pregnancy / blood vessel remodeling / blood vessel diameter maintenance / oxygen binding / pyridoxal phosphate binding / cellular response to hypoxia / ubiquitin protein ligase binding / heme binding / negative regulation of apoptotic process / enzyme binding / protein homodimerization activity / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Cystathionine beta-synthase, C-terminal domain / Cystathionine beta-synthase / Cysteine synthase/cystathionine beta-synthase, pyridoxal-phosphate attachment site / Cysteine synthase/cystathionine beta-synthase P-phosphate attachment site. / CBS domain superfamily / Domain in cystathionine beta-synthase and other proteins. / Pyridoxal-phosphate dependent enzyme / Tryptophan synthase beta subunit-like PLP-dependent enzyme / Pyridoxal-phosphate dependent enzyme / CBS domain ...Cystathionine beta-synthase, C-terminal domain / Cystathionine beta-synthase / Cysteine synthase/cystathionine beta-synthase, pyridoxal-phosphate attachment site / Cysteine synthase/cystathionine beta-synthase P-phosphate attachment site. / CBS domain superfamily / Domain in cystathionine beta-synthase and other proteins. / Pyridoxal-phosphate dependent enzyme / Tryptophan synthase beta subunit-like PLP-dependent enzyme / Pyridoxal-phosphate dependent enzyme / CBS domain / CBS domain / CBS domain profile.
Similarity search - Domain/homology
Cystathionine beta-synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsMcCorvie TJ / Yue WW
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Architecture and regulation of filamentous human cystathionine beta-synthase.
Authors: Thomas J McCorvie / Douglas Adamoski / Raquel A C Machado / Jiazhi Tang / Henry J Bailey / Douglas S M Ferreira / Claire Strain-Damerell / Arnaud Baslé / Andre L B Ambrosio / Sandra M G Dias / Wyatt W Yue /
Abstract: Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic ...Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.
History
DepositionFeb 23, 2024-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19742.png

Downloads & links

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Map

FileDownload / File: emd_19742.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 360 pix.
= 381.6 Å		1.06 Å/pix.
x 360 pix.
= 381.6 Å		1.06 Å/pix.
x 360 pix.
= 381.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.29
Minimum - Maximum-1.6166804 - 2.3833497
Average (Standard dev.)0.00072038145 (±0.0376517)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 381.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19742_msk_1.map
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Additional map: Locally sharpened map

Fileemd_19742_additional_1.map
AnnotationLocally sharpened map
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Half map: #1

Fileemd_19742_half_map_1.map
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Half map: #2

Fileemd_19742_half_map_2.map
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Sample components

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Entire : Helical assembly of full-length human cystathionine beta-synthase...

EntireName: Helical assembly of full-length human cystathionine beta-synthase bound to SAM in the activated state
Components
  • Complex: Helical assembly of full-length human cystathionine beta-synthase bound to SAM in the activated state
    • Protein or peptide: cystathionine beta-synthaseCystathionine beta synthase

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Supramolecule #1: Helical assembly of full-length human cystathionine beta-synthase...

SupramoleculeName: Helical assembly of full-length human cystathionine beta-synthase bound to SAM in the activated state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: cystathionine beta-synthase

MacromoleculeName: cystathionine beta-synthase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: cystathionine beta-synthase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPSETPQAEV GPTGCPHRSG PHSAKGSLEK GSPEDKEAKE PLWIRPDAPS RCTWQLGRPA SESPHHHTA PAKSPKILPD ILKKIGDTPM VRINKIGKKF GLKCELLAKC EFFNAGGSVK D RISLRMIE DAERDGTLKP GDTIIEPTSG NTGIGLALAA AVRGYRCIIV ...String:
MPSETPQAEV GPTGCPHRSG PHSAKGSLEK GSPEDKEAKE PLWIRPDAPS RCTWQLGRPA SESPHHHTA PAKSPKILPD ILKKIGDTPM VRINKIGKKF GLKCELLAKC EFFNAGGSVK D RISLRMIE DAERDGTLKP GDTIIEPTSG NTGIGLALAA AVRGYRCIIV MPEKMSSEKV DV LRALGAE IVRTPTNARF DSPESHVGVA WRLKNEIPNS HILDQYRNAS NPLAHYDTTA DEI LQQCDG KLDMLVASVG TGGTITGIAR KLKEKCPGCR IIGVDPEGSI LAEPEELNQT EQTT YEVEG IGYDFIPTVL DRTVVDKWFK SNDEEAFTFA RMLIAQEGLL CGGSAGSTVA VAVKA AQEL QEGQRCVVIL PDSVRNYMTK FLSDRWMLQK GFLKEEDLTE KKPWWWHLRV QELGLS APL TVLPTITCGH TIEILREKGF DQAPVVDEAG VILGMVTLGN MLSSLLAGKV QPSDQVG KV IYKQFKQIRL TDTLGRLSHI LEMDHFALVV HEQIQYHSTG KSSQRQMVFG VVTAIDLL N FVAAQERDQK AAHHHHHH

UniProtKB: Cystathionine beta-synthase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMHEPES
200.0 mMsodium chlorideNaClSodium chloride
2.0 mMTCEP
0.005 % (v/v)Tween-20
5.0 mMS-adenosyl-l-methionineS-Adenosyl methionine

Details: filter sterile 25 mM HEPES, pH 7.5, 200 mM NaCl, 2.0 mM TCEP, 0.005% (v/v) tween-20, 5 mM SAM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 11220 / Average exposure time: 3.53 sec. / Average electron dose: 39.96 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5240414
Startup modelType of model: OTHER / Details: ab-inito from data
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.1.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.1.0)
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.0)
Details: Locally masked refinement from helical refinement using soft mask without enforcing helical symmetry.
Number images used: 425260
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainPDB ID
source_name: AlphaFold, initial_model_type: in silico model
source_name: PDB, initial_model_type: experimental model

4uuu

source_name: PDB, initial_model_type: experimental model

4pcu

RefinementProtocol: FLEXIBLE FIT

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