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- EMDB-13802: Structure of full-length, dimeric, soluble somatic angiotensin I-... -

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Basic information

Entry
Database: EMDB / ID: EMD-13802
TitleStructure of full-length, dimeric, soluble somatic angiotensin I-converting enzyme
Map dataGlobally-sharpened map of full-length dimeric somatic angiotensin I-converting enzyme showing interacting N-domains and disordered C-domains
Sample
  • Complex: Full-length, soluble, dimeric somatic angiotensin I-converting enzyme
    • Protein or peptide: Angiotensin I-converting enzyme
KeywordsZinc metalloprotease Dicarboxypeptidase Glycoprotein / HYDROLASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.79 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-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13802.png

Downloads & links

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Map

FileDownload / File: emd_13802.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally-sharpened map of full-length dimeric somatic angiotensin I-converting enzyme showing interacting N-domains and disordered C-domains
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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.65
Minimum - Maximum-1.6559213 - 3.2416894
Average (Standard dev.)0.0015133391 (±0.054605976)
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_13802_msk_1.map
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Mask #2

Fileemd_13802_msk_2.map
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Additional map: Raw, unfiltered full map from non-uniform refinement of...

Fileemd_13802_additional_1.map
AnnotationRaw, unfiltered full map from non-uniform refinement of full-length dimeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

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Histogram

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

Fileemd_13802_half_map_1.map
AnnotationRaw, unfiltered half-map A of full-length dimeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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

Fileemd_13802_half_map_2.map
AnnotationRaw, unfiltered half-map B of full-length dimeric somatic angiotensin I-converting enzyme
Projections & Slices
AxesZYX

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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 I-converting enzyme

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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: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 279 KDa

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Macromolecule #1: Angiotensin I-converting enzyme

MacromoleculeName: Angiotensin I-converting enzyme / type: protein_or_peptide / ID: 1
Details: Soluble secreted form of human somatic angiotensin I-converting enzyme terminating at Ser1211
Enantiomer: LEVO / EC number: peptidyl-dipeptidase A
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: LDPGLQPGNF SADEAGAQLF AQSYNSSAEQ V LFQSVAAS WAHDTNITAE NARRQEEAAL LSQEFAEAWG QKAKELYEPI WQNFTDPQLR RI IGAVRTL GSANLPLAKR QQYNALLSNM SRIYSTAKVC LPNKTATCWS LDPDLTNILA SSR SYAMLL FAWEGWHNAA ...String:
LDPGLQPGNF SADEAGAQLF AQSYNSSAEQ V LFQSVAAS WAHDTNITAE NARRQEEAAL LSQEFAEAWG QKAKELYEPI WQNFTDPQLR RI IGAVRTL GSANLPLAKR QQYNALLSNM SRIYSTAKVC LPNKTATCWS LDPDLTNILA SSR SYAMLL FAWEGWHNAA GIPLKPLYED FTALSNEAYK QDGFTDTGAY WRSWYNSPTF EDDL EHLYQ QLEPLYLNLH AFVRRALHRR YGDRYINLRG PIPAHLLGDM WAQSWENIYD MVVPF PDKP NLDVTSTMLQ QGWNATHMFR VAEEFFTSLE LSPMPPEFWE GSMLEKPADG REVVCH ASA WDFYNRKDFR IKQCTRVTMD QLSTVHHEMG HIQYYLQYKD LPVSLRRGAN PGFHEAI GD VLALSVSTPE HLHKIGLLDR VTNDTESDIN YLLKMALEKI AFLPFGYLVD QWRWGVFS G RTPPSRYNFD WWYLRTKYQG ICPPVTRNET HFDAGAKFHV PNVTPYIRYF VSFVLQFQF HEALCKEAGY EGPLHQCDIY RSTKAGAKLR KVLQAGSSRP WQEVLKDMVG LDALDAQPLL KYFQLVTQW LQEQNQQNGE VLGWPEYQWH PPLPDNYPEG IDLVTDEAEA SKFVEEYDRT S QVVWNEYA EANWNYNTNI TTETSKILLQ KNMQIANHTL KYGTQARKFD VNQLQNTTIK RI IKKVQDL ERAALPAQEL EEYNKILLDM ETTYSVATVC HPNGSCLQLE PDLTNVMATS RKY EDLLWA WEGWRDKAGR AILQFYPKYV ELINQAARLN GYVDAGDSWR SMYETPSLEQ DLER LFQEL QPLYLNLHAY VRRALHRHYG AQHINLEGPI PAHLLGNMWA QTWSNIYDLV VPFPS APSM DTTEAMLKQG WTPRRMFKEA DDFFTSLGLL PVPPEFWNKS MLEKPTDGRE VVCHAS AWD FYNGKDFRIK QCTTVNLEDL VVAHHEMGHI QYFMQYKDLP VALREGANPG FHEAIGD VL ALSVSTPKHL HSLNLLSSEG GSDEHDINFL MKMALDKIAF IPFSYLVDQW RWRVFDGS I TKENYNQEWW SLRLKYQGLC PPVPRTQGDF DPGAKFHIPS SVPYIRYFVS FIIQFQFHE ALCQAAGHTG PLHKCDIYQS KEAGQRLATA MKLGFSRPWP EAMQLITGQP NMSASAMLSY FKPLLDWLR TENELHGEKL GWPQYNWTPN SARSEGPLPD S

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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 / 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
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
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: 100 / Software - Name: cryoSPARC (ver. 3.2)
Details: 70 dimeric classes were selected from 2D classification for non-uniform refinement in cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionNumber classes used: 70 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Details: Non-uniform refinement of the full dimer / Number images used: 123059
FSC plot (resolution estimation)

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Atomic model buiding 1

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

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