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- EMDB-13801: Local refinement structure of the C-domain of full-length, monome... -

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Entry
Database: EMDB / ID: EMD-13801
TitleLocal refinement structure of the C-domain of full-length, monomeric, soluble somatic angiotensin I-converting enzyme
Map dataGlobally-sharpened map from local refinement of the C-domain of full-length monomeric somatic angiotensin I-converting enzyme
Sample
  • Complex: Full-length, soluble, monomeric somatic angiotensin I-converting enzyme
    • Protein or peptide: Angiotensin-converting enzyme
  • Ligand: CHLORIDE IONChloride
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


mononuclear cell proliferation / cell proliferation in bone marrow / tripeptidyl-peptidase activity / bradykinin receptor binding / regulation of angiotensin metabolic process / exopeptidase activity / substance P catabolic process / response to laminar fluid shear stress / peptidyl-dipeptidase A / regulation of renal output by angiotensin ...mononuclear cell proliferation / cell proliferation in bone marrow / tripeptidyl-peptidase activity / bradykinin receptor binding / regulation of angiotensin metabolic process / exopeptidase activity / substance P catabolic process / response to laminar fluid shear stress / peptidyl-dipeptidase A / regulation of renal output by angiotensin / negative regulation of calcium ion import / positive regulation of peptidyl-cysteine S-nitrosylation / positive regulation of systemic arterial blood pressure / negative regulation of gap junction assembly / metallodipeptidase activity / cellular response to aldosterone / hormone catabolic process / angiogenesis involved in coronary vascular morphogenesis / bradykinin catabolic process / response to thyroid hormone / negative regulation of glucose import / vasoconstriction / neutrophil mediated immunity / hormone metabolic process / regulation of hematopoietic stem cell proliferation / regulation of smooth muscle cell migration / antigen processing and presentation of peptide antigen via MHC class I / embryo development ending in birth or egg hatching / mitogen-activated protein kinase binding / chloride ion binding / positive regulation of neurogenesis / mitogen-activated protein kinase kinase binding / eating behavior / arachidonic acid secretion / post-transcriptional regulation of gene expression / lung alveolus development / peptide catabolic process / heterocyclic compound binding / heart contraction / 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 / angiotensin maturation / hematopoietic stem cell differentiation / blood vessel remodeling / Metabolism of Angiotensinogen to Angiotensins / animal organ regeneration / amyloid-beta metabolic process / carboxypeptidase activity / positive regulation of vasoconstriction / sperm midpiece / blood vessel diameter maintenance / response to nutrient levels / basal plasma membrane / kidney development / female pregnancy / angiotensin-activated signaling pathway / cellular response to glucose stimulus / brush border membrane / regulation of synaptic plasticity / metalloendopeptidase activity / regulation of blood pressure / male gonad development / metallopeptidase activity / actin binding / peptidase activity / spermatogenesis / endopeptidase activity / response to lipopolysaccharide / lysosome / response to hypoxia / calmodulin binding / endosome / response to xenobiotic stimulus / positive regulation of apoptotic process / external side of plasma membrane / negative regulation of gene expression / proteolysis / extracellular space / extracellular exosome / zinc ion binding / 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: 4.08 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 30, 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_13801.png

Downloads & links

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Map

FileDownload / File: emd_13801.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally-sharpened map from local refinement of the C-domain of full-length monomeric somatic angiotensin I-converting enzyme
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-2.8397968 - 3.1996722
Average (Standard dev.)0.0030847029 (±0.06288382)
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_13801_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Mask #2

Fileemd_13801_msk_2.map
Projections & Slices
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Additional map: Density-modified map (Phenix resolve cryo-EM) from local refinement...

Fileemd_13801_additional_1.map
AnnotationDensity-modified map (Phenix resolve cryo-EM) from local refinement of the C-domain of full-length monomeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw, unfiltered full map from local refinement of...

Fileemd_13801_additional_2.map
AnnotationRaw, unfiltered full map from local refinement of the C-domain of full-length monomeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Raw, unfiltered half-map B from local refinement of...

Fileemd_13801_half_map_1.map
AnnotationRaw, unfiltered half-map B from local refinement of the C-domain of full-length monomeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Raw, unfiltered half-map A from local refinement of...

Fileemd_13801_half_map_2.map
AnnotationRaw, unfiltered half-map A from local refinement of the C-domain 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: CHLORIDE IONChloride
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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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,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 #4: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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 chloridesodium 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
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
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
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1)
Details: 1 class was selected from focused 3D classification without alignment in RELION for C-domain local refinement in cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.08 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Details: Local refinement with a mask around the C-domain / Number images used: 42701
FSC plot (resolution estimation)

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Atomic model buiding 1

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

PDB-7q4c:
Local refinement structure of the C-domain of full-length, monomeric, soluble somatic angiotensin I-converting enzyme

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