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- EMDB-13804: Local refinement structure of a single N-domain of full-length, d... -

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Entry
Database: EMDB / ID: EMD-13804
TitleLocal refinement structure of a single N-domain of full-length, dimeric, soluble somatic angiotensin I-converting enzyme
Map dataGlobally-sharpened map from local refinement of a single N-domain of full-length dimeric somatic angiotensin I-converting enzyme
Sample
  • Complex: Full-length, soluble, dimeric somatic angiotensin I-converting enzyme
    • Protein or peptide: Angiotensin-converting enzyme
  • Ligand: ZINC ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsZinc metalloprotease Dicarboxypeptidase Glycoprotein / HYDROLASE
Function / homology
Function and homology information


mononuclear cell proliferation / cell proliferation in bone marrow / bradykinin receptor binding / exopeptidase activity / regulation of angiotensin metabolic process / substance P catabolic process / tripeptidyl-peptidase activity / peptidyl-dipeptidase A / regulation of renal output by angiotensin / negative regulation of gap junction assembly ...mononuclear cell proliferation / cell proliferation in bone marrow / bradykinin receptor binding / exopeptidase activity / regulation of angiotensin metabolic process / substance P catabolic process / tripeptidyl-peptidase activity / peptidyl-dipeptidase A / regulation of renal output by angiotensin / negative regulation of gap junction assembly / hormone catabolic process / bradykinin catabolic process / metallodipeptidase activity / regulation of smooth muscle cell migration / regulation of hematopoietic stem cell proliferation / neutrophil mediated immunity / hormone metabolic process / mitogen-activated protein kinase binding / mitogen-activated protein kinase kinase binding / chloride ion binding / arachidonate secretion / post-transcriptional regulation of gene expression / peptide catabolic process / heart contraction / antigen processing and presentation of peptide antigen via MHC class I / positive regulation of systemic arterial blood pressure / regulation of heart rate by cardiac conduction / regulation of systemic arterial blood pressure by renin-angiotensin / blood vessel remodeling / amyloid-beta metabolic process / hematopoietic stem cell differentiation / peptidyl-dipeptidase activity / regulation of vasoconstriction / Metabolism of Angiotensinogen to Angiotensins / angiotensin maturation / metallocarboxypeptidase activity / blood vessel diameter maintenance / angiotensin-activated signaling pathway / kidney development / regulation of synaptic plasticity / metalloendopeptidase activity / regulation of blood pressure / male gonad development / metallopeptidase activity / peptidase activity / actin binding / spermatogenesis / endopeptidase activity / calmodulin binding / lysosome / endosome / negative regulation of gene expression / external side of plasma membrane / proteolysis / extracellular space / extracellular exosome / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
Peptidase M2, peptidyl-dipeptidase A / Angiotensin-converting enzyme / Peptidase family M2 domain profile. / 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: 3.63 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-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13804.png

Downloads & links

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Map

FileDownload / File: emd_13804.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally-sharpened map from local refinement of a single N-domain of full-length dimeric somatic angiotensin I-converting enzyme
Projections & slices

Image control

Size
Brightness
Contrast
Others
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: 0.6
Minimum - Maximum-1.668471 - 2.7439525
Average (Standard dev.)0.00065238104 (±0.042877566)
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_13804_msk_1.map
Projections & Slices
AxesZYX

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Mask #2

Fileemd_13804_msk_2.map
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Additional map: Raw, unfiltered full map from local refinement of...

Fileemd_13804_additional_1.map
AnnotationRaw, unfiltered full map from local refinement of a single N-domain of full-length dimeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_13804_additional_2.map
AnnotationDensity-modified map (Phenix resolve cryo-EM) from local refinement of a single N-domain of full-length dimeric 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 from local refinement of...

Fileemd_13804_half_map_1.map
AnnotationRaw, unfiltered half-map A from local refinement of a single N-domain of full-length dimeric 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_13804_half_map_2.map
AnnotationRaw, unfiltered half-map B from local refinement of a single N-domain of full-length dimeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Full-length, soluble, dimeric somatic angiotensin I-converting enzyme

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

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Supramolecule #1: Full-length, soluble, dimeric somatic angiotensin I-converting enzyme

SupramoleculeName: Full-length, soluble, dimeric somatic angiotensin I-converting enzyme
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 279 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

UniProtKB: Angiotensin-converting enzyme

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

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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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 / Pretreatment - Time: 30 sec.
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
Startup modelType of model: OTHER / Details: Ab initio 3D model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2)
Details: Local refinement (C1) with a mask around a single N-domain following C-domain particle subtraction in RELION, C2 symmetry expansion in cryoSPARC, and local refinement of the two interacting N-domains
Number images used: 215632
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 3.2)
Details: 1 dimeric class was selected from focused 3D classification without alignment in RELION for local 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-7q4e:
Local refinement structure of a single N-domain of full-length, dimeric, soluble somatic angiotensin I-converting enzyme

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