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- EMDB-13797: Structure of full-length, monomeric, soluble somatic angiotensin ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13797
TitleStructure of full-length, monomeric, soluble somatic angiotensin I-converting enzyme showing the N- and C-terminal ellipsoid domains
Map dataGlobally-sharpened map of full-length monomeric somatic angiotensin I-converting enzyme showing N-terminal and C-terminal ellipsoid domains
Sample
  • Complex: full-length, soluble, monomeric somatic angiotensin I-converting enzyme
    • Protein or peptide: Angiotensin-converting enzyme
  • Ligand: ZINC ION
  • Ligand: CHLORIDE ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
Function / homology
Function and homology information


mononuclear cell proliferation / cell proliferation in bone marrow / bradykinin receptor binding / regulation of angiotensin metabolic process / exopeptidase activity / substance P catabolic process / peptidyl-dipeptidase A / tripeptidyl-peptidase activity / regulation of renal output by angiotensin / negative regulation of calcium ion import ...mononuclear cell proliferation / cell proliferation in bone marrow / bradykinin receptor binding / regulation of angiotensin metabolic process / exopeptidase activity / substance P catabolic process / peptidyl-dipeptidase A / tripeptidyl-peptidase activity / regulation of renal output by angiotensin / negative regulation of calcium ion import / positive regulation of peptidyl-cysteine S-nitrosylation / response to laminar fluid shear stress / negative regulation of gap junction assembly / metallodipeptidase activity / positive regulation of systemic arterial blood pressure / cellular response to aldosterone / hormone catabolic process / bradykinin catabolic process / angiogenesis involved in coronary vascular morphogenesis / response to thyroid hormone / antigen processing and presentation of peptide antigen via MHC class I / negative regulation of glucose import / vasoconstriction / hormone metabolic process / neutrophil mediated immunity / regulation of smooth muscle cell migration / regulation of hematopoietic stem cell proliferation / mitogen-activated protein kinase binding / embryo development ending in birth or egg hatching / chloride ion binding / mitogen-activated protein kinase kinase binding / positive regulation of neurogenesis / arachidonic acid secretion / post-transcriptional regulation of gene expression / eating behavior / heterocyclic compound binding / lung alveolus development / heart contraction / peptide catabolic process / response to dexamethasone / regulation of heart rate by cardiac conduction / regulation of systemic arterial blood pressure by renin-angiotensin / regulation of vasoconstriction / peptidyl-dipeptidase activity / hematopoietic stem cell differentiation / blood vessel remodeling / angiotensin maturation / amyloid-beta metabolic process / animal organ regeneration / Metabolism of Angiotensinogen to Angiotensins / carboxypeptidase activity / positive regulation of vasoconstriction / sperm midpiece / blood vessel diameter maintenance / response to nutrient levels / basal plasma membrane / kidney development / angiotensin-activated signaling pathway / female pregnancy / cellular response to glucose stimulus / brush border membrane / regulation of synaptic plasticity / metalloendopeptidase activity / regulation of blood pressure / metallopeptidase activity / male gonad development / peptidase activity / actin binding / spermatogenesis / endopeptidase activity / response to lipopolysaccharide / lysosome / calmodulin binding / response to hypoxia / endosome / response to xenobiotic stimulus / positive regulation of apoptotic process / external side of plasma membrane / negative regulation of gene expression / proteolysis / extracellular space / zinc ion binding / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Peptidase family M2 domain profile. / Peptidase M2, peptidyl-dipeptidase A / Angiotensin-converting enzyme / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Angiotensin-converting enzyme
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.34 Å
AuthorsLubbe L / Sewell BT / Sturrock ED
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)ST/R002754/1 United Kingdom
CitationJournal: EMBO J / Year: 2022
Title: Cryo-EM reveals mechanisms of angiotensin I-converting enzyme allostery and dimerization.
Authors: Lizelle Lubbe / Bryan Trevor Sewell / Jeremy D Woodward / Edward D Sturrock /
Abstract: Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I-converting enzyme (sACE) ...Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I-converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X-ray crystallography and molecular dynamics simulations. Here, we report the first cryo-EM structures of full-length, glycosylated, soluble sACE (sACE ). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N- and C-terminal domains of monomeric sACE were resolved at 3.7 and 4.1 Å, respectively, while the interacting N-terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.
History
DepositionOct 29, 2021-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13797.png

Downloads & links

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Map

FileDownload / File: emd_13797.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally-sharpened map of full-length monomeric somatic angiotensin I-converting enzyme showing N-terminal and C-terminal ellipsoid domains
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.2264988 - 1.9009315
Average (Standard dev.)0.0032340405 (±0.047989495)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13797_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_13797_msk_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Additional map: Raw, unfiltered full map of full-length monomeric somatic...

Fileemd_13797_additional_1.map
AnnotationRaw, unfiltered full map of full-length monomeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Raw, unfiltered half-map B of full-length monomeric somatic...

Fileemd_13797_half_map_1.map
AnnotationRaw, unfiltered half-map B of full-length monomeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Raw, unfiltered half-map A of full-length monomeric somatic...

Fileemd_13797_half_map_2.map
AnnotationRaw, unfiltered half-map A of full-length monomeric somatic angiotensin I-converting enzyme
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 : full-length, soluble, monomeric somatic angiotensin I-converting ...

EntireName: full-length, soluble, monomeric somatic angiotensin I-converting enzyme
Components
  • Complex: full-length, soluble, monomeric somatic angiotensin I-converting enzyme
    • Protein or peptide: Angiotensin-converting enzyme
  • Ligand: ZINC ION
  • Ligand: CHLORIDE ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: full-length, soluble, monomeric somatic angiotensin I-converting ...

SupramoleculeName: full-length, soluble, monomeric somatic angiotensin I-converting enzyme
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant plasmid: pcDNA3.1+
Molecular weightTheoretical: 139 KDa

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Macromolecule #1: Angiotensin-converting enzyme

MacromoleculeName: Angiotensin-converting enzyme / type: protein_or_peptide / ID: 1
Details: Soluble secreted form of human somatic angiotensin I-converting enzyme terminating at Ser1211
Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 139.614 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: LDPGLQPGNF SADEAGAQLF AQSYNSSAEQ VLFQSVAASW AHDTNITAEN ARRQEEAALL SQEFAEAWGQ KAKELYEPIW QNFTDPQLR RIIGAVRTLG SANLPLAKRQ QYNALLSNMS RIYSTAKVCL PNKTATCWSL DPDLTNILAS SRSYAMLLFA W EGWHNAAG ...String:
LDPGLQPGNF SADEAGAQLF AQSYNSSAEQ VLFQSVAASW AHDTNITAEN ARRQEEAALL SQEFAEAWGQ KAKELYEPIW QNFTDPQLR RIIGAVRTLG SANLPLAKRQ QYNALLSNMS RIYSTAKVCL PNKTATCWSL DPDLTNILAS SRSYAMLLFA W EGWHNAAG IPLKPLYEDF TALSNEAYKQ DGFTDTGAYW RSWYNSPTFE DDLEHLYQQL EPLYLNLHAF VRRALHRRYG DR YINLRGP IPAHLLGDMW AQSWENIYDM VVPFPDKPNL DVTSTMLQQG WNATHMFRVA EEFFTSLELS PMPPEFWEGS MLE KPADGR EVVCHASAWD FYNRKDFRIK QCTRVTMDQL STVHHEMGHI QYYLQYKDLP VSLRRGANPG FHEAIGDVLA LSVS TPEHL HKIGLLDRVT NDTESDINYL LKMALEKIAF LPFGYLVDQW RWGVFSGRTP PSRYNFDWWY LRTKYQGICP PVTRN ETHF DAGAKFHVPN VTPYIRYFVS FVLQFQFHEA LCKEAGYEGP LHQCDIYRST KAGAKLRKVL QAGSSRPWQE VLKDMV GLD ALDAQPLLKY FQLVTQWLQE QNQQNGEVLG WPEYQWHPPL PDNYPEGIDL VTDEAEASKF VEEYDRTSQV VWNEYAE AN WNYNTNITTE TSKILLQKNM QIANHTLKYG TQARKFDVNQ LQNTTIKRII KKVQDLERAA LPAQELEEYN KILLDMET T YSVATVCHPN GSCLQLEPDL TNVMATSRKY EDLLWAWEGW RDKAGRAILQ FYPKYVELIN QAARLNGYVD AGDSWRSMY ETPSLEQDLE RLFQELQPLY LNLHAYVRRA LHRHYGAQHI NLEGPIPAHL LGNMWAQTWS NIYDLVVPFP SAPSMDTTEA MLKQGWTPR RMFKEADDFF TSLGLLPVPP EFWNKSMLEK PTDGREVVCH ASAWDFYNGK DFRIKQCTTV NLEDLVVAHH E MGHIQYFM QYKDLPVALR EGANPGFHEA IGDVLALSVS TPKHLHSLNL LSSEGGSDEH DINFLMKMAL DKIAFIPFSY LV DQWRWRV FDGSITKENY NQEWWSLRLK YQGLCPPVPR TQGDFDPGAK FHIPSSVPYI RYFVSFIIQF QFHEALCQAA GHT GPLHKC DIYQSKEAGQ RLATAMKLGF SRPWPEAMQL ITGQPNMSAS AMLSYFKPLL DWLRTENELH GEKLGWPQYN WTPN SARSE GPLPDS

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #9: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 9 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #10: water

MacromoleculeName: water / type: ligand / ID: 10 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
125.0 mMNaClsodium chloride
0.01 mMZnCl2zinc chloride
50.0 mMC8H18N2O4SHEPES

Details: Solutions were prepared with deionized water
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Diluted protein (in buffer containing zinc chloride and sodium chloride) was incubated on ice for 30 minutes after which it was applied to the grid, incubated for 30 seconds, and blotted for ...Details: Diluted protein (in buffer containing zinc chloride and sodium chloride) was incubated on ice for 30 minutes after which it was applied to the grid, incubated for 30 seconds, and blotted for 3 seconds before plunging.
DetailsThe protein was stored at 3.0mg/ml in 50mM HEPES (pH 7.5) and diluted immediately prior to grid preparation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 11628 / Average exposure time: 3.0 sec. / Average electron dose: 43.0 e/Å2
Details: Images were recorded in super-resolution mode with 40 frames per image
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1727045
Details: Topaz-denoising was performed on all curated micrographs, a Topaz model trained on this dataset, and used for picking
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2) / Software - details: patch-based CTF estimation / Details: cryoSPARC patch-based CTF estimation
Startup modelType of model: OTHER / Details: Ab initio 3D model
Final reconstructionNumber classes used: 81 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Details: cryoSPARC non-uniform refinement was used / Number images used: 121372
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 100 / Software - Name: cryoSPARC (ver. 3.2)
Details: 81 classes were selected for 3D non-uniform refinement in cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-7q3y:
Structure of full-length, monomeric, soluble somatic angiotensin I-converting enzyme showing the N- and C-terminal ellipsoid domains

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