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- PDB-8s5m: Full-length human cystathionine beta-synthase with C-terminal 6xH... -

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Basic information

Entry
Database: PDB / ID: 8s5m
TitleFull-length human cystathionine beta-synthase with C-terminal 6xHis-tag, SAM bound, activated state, helical reconstruction
ComponentsCystathionine beta-synthase
KeywordsTRANSFERASE / Filament / Allostery
Function / homology
Function and homology information


Cysteine formation from homocysteine / homocysteine catabolic process / modified amino acid binding / cystathionine beta-synthase / cysteine biosynthetic process via cystathionine / cystathionine beta-synthase activity / Metabolism of ingested SeMet, Sec, MeSec into H2Se / homocysteine metabolic process / hydrogen sulfide biosynthetic process / carbon monoxide binding ...Cysteine formation from homocysteine / homocysteine catabolic process / modified amino acid binding / cystathionine beta-synthase / cysteine biosynthetic process via cystathionine / cystathionine beta-synthase activity / Metabolism of ingested SeMet, Sec, MeSec into H2Se / homocysteine metabolic process / hydrogen sulfide biosynthetic process / carbon monoxide binding / L-serine catabolic process / L-serine metabolic process / cartilage development involved in endochondral bone morphogenesis / regulation of nitric oxide mediated signal transduction / cysteine biosynthetic process / L-cysteine catabolic process / cerebellum morphogenesis / nitric oxide binding / transsulfuration / DNA protection / cysteine biosynthetic process from serine / endochondral ossification / S-adenosyl-L-methionine binding / response to folic acid / nitrite reductase (NO-forming) activity / superoxide metabolic process / blood vessel remodeling / maternal process involved in female pregnancy / 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 / nucleus / metal ion binding / 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. / : / 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 superfamily ...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. / : / 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 superfamily / CBS domain / CBS domain / CBS domain profile.
Similarity search - Domain/homology
S-ADENOSYLMETHIONINE / Cystathionine beta-synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 Å
AuthorsMcCorvie, T.J. / Yue, W.W.
Funding support United Kingdom, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cystathionine beta-synthase
B: Cystathionine beta-synthase
C: Cystathionine beta-synthase
D: Cystathionine beta-synthase
E: Cystathionine beta-synthase
F: Cystathionine beta-synthase
G: Cystathionine beta-synthase
H: Cystathionine beta-synthase
I: Cystathionine beta-synthase
J: Cystathionine beta-synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)620,34920
Polymers616,36410
Non-polymers3,98410
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Cystathionine beta-synthase / Beta-thionase / Serine sulfhydrase


Mass: 61636.426 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CBS / Production host: Escherichia coli (E. coli) / References: UniProt: P35520, cystathionine beta-synthase
#2: Chemical
ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Helical assembly of full-length human cystathionine beta-synthase in the basal state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2200 mMSodium chlorideNaCl1
32 mMTCEP1
40.005 % (v/v)Tween-201
55 mMS-adenosyl-l-methionine1
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 900 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.53 sec. / Electron dose: 39.96 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11220
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.1.0particle selection
4cryoSPARC3.1.0CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCab-inito from datainitial Euler assignment
10cryoSPARCab-inito from datafinal Euler assignment
11cryoSPARC3.1.0classification
12cryoSPARC3.1.03D reconstruction
13PHENIXmodel refinement
14ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -178.6 ° / Axial rise/subunit: 46.7 Å / Axial symmetry: D2
Particle selectionNum. of particles selected: 5240414
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 425260 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingB value: 79.15 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDSource nameTypeAccession codeInitial refinement model-ID
11AlphaFoldin silico model
24UUU

4uuu

1PDBexperimental model4UUU2
34PCU

4pcu

1PDBexperimental model4PCU3
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 79.01 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003911870
ELECTRON MICROSCOPYf_angle_d0.792316120
ELECTRON MICROSCOPYf_chiral_restr0.04591890
ELECTRON MICROSCOPYf_plane_restr0.00412000
ELECTRON MICROSCOPYf_dihedral_angle_d12.6151660

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