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- EMDB-14389: Structure of Mycobacterium tuberculosis adenylyl cyclase Rv1625c / Cya -

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Basic information

Entry
Database: EMDB / ID: EMD-14389
TitleStructure of Mycobacterium tuberculosis adenylyl cyclase Rv1625c / Cya
Map data
Sample
  • Complex: A complex of Rv1625c / Cya homodimer bound to nano body Nb4
    • Complex: Adenylate cyclase
      • Protein or peptide: Adenylate cyclase
    • Complex: Nanobody Nb4
      • Protein or peptide: Nanobody Nb4
  • Ligand: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MANGANESE (II) ION
KeywordsAdenylyl cyclase / membrane protein / cyclic adenosine monophosphate / signal transduction / nanobody
Function / homology
Function and homology information


cyclic nucleotide biosynthetic process / adenylate cyclase activity / membrane => GO:0016020 / intracellular signal transduction
Similarity search - Function
Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / Adenylyl- / guanylyl cyclase, catalytic domain / Adenylate and Guanylate cyclase catalytic domain / Adenylyl cyclase class-3/4/guanylyl cyclase / Guanylate cyclase domain profile. / Nucleotide cyclase
Similarity search - Domain/homology
Biological speciesMycobacterium tuberculosis (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsMehta V / Khanppnavar B
Funding support Switzerland, 3 items
OrganizationGrant numberCountry
Swiss National Science Foundation150665 Switzerland
Swiss National Science Foundation176992 Switzerland
Swiss National Science Foundation184951 Switzerland
CitationJournal: Elife / Year: 2022
Title: Structure of Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases.
Authors: Ved Mehta / Basavraj Khanppnavar / Dina Schuster / Ilayda Kantarci / Irene Vercellino / Angela Kosturanova / Tarun Iype / Sasa Stefanic / Paola Picotti / Volodymyr M Korkhov /
Abstract: adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular ... adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.
History
DepositionFeb 20, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14389.png

Downloads & links

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Map

FileDownload / File: emd_14389.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 400 pix.
= 264. Å		0.66 Å/pix.
x 400 pix.
= 264. Å		0.66 Å/pix.
x 400 pix.
= 264. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.009
Minimum - Maximum-0.04471881 - 0.06185481
Average (Standard dev.)0.00004929449 (±0.0011608645)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_14389_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14389_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : A complex of Rv1625c / Cya homodimer bound to nano body Nb4

EntireName: A complex of Rv1625c / Cya homodimer bound to nano body Nb4
Components
  • Complex: A complex of Rv1625c / Cya homodimer bound to nano body Nb4
    • Complex: Adenylate cyclase
      • Protein or peptide: Adenylate cyclase
    • Complex: Nanobody Nb4
      • Protein or peptide: Nanobody Nb4
  • Ligand: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: A complex of Rv1625c / Cya homodimer bound to nano body Nb4

SupramoleculeName: A complex of Rv1625c / Cya homodimer bound to nano body Nb4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Adenylate cyclase

SupramoleculeName: Adenylate cyclase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Supramolecule #3: Nanobody Nb4

SupramoleculeName: Nanobody Nb4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Vicugna pacos (alpaca)

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Macromolecule #1: Adenylate cyclase

MacromoleculeName: Adenylate cyclase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: adenylate cyclase
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 50.386613 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSAWSHPQF EKGSGLEVLF QGPSGHMAAR KCGAPPIAAD GSTRRPDCVT AVRTQARAPT QHYAESVARR QRVLTITAWL AVVVTGSFA LMQLATGAGG WYIALINVFT AVTFAIVPLL HRFGGLVAPL TFIGTAYVAI FAIGWDVGTD AGAQFFFLVA A ALVVLLVG ...String:
MGSAWSHPQF EKGSGLEVLF QGPSGHMAAR KCGAPPIAAD GSTRRPDCVT AVRTQARAPT QHYAESVARR QRVLTITAWL AVVVTGSFA LMQLATGAGG WYIALINVFT AVTFAIVPLL HRFGGLVAPL TFIGTAYVAI FAIGWDVGTD AGAQFFFLVA A ALVVLLVG IEHTALAVGL AAVAAGLVIA LEFLVPPDTG LQPPWAMSVS FVLTTVSACG VAVATVWFAL RDTARAEAVM EA EHDRSEA LLANMLPASI AERLKEPERN IIADKYDEAS VLFADIVGFT ERASSTAPAD LVRFLDRLYS AFDELVDQHG LEK IKVSGD SYMVVSGVPR PRPDHTQALA DFALDMTNVA AQLKDPRGNP VPLRVGLATG PVVAGVVGSR RFFYDVWGDA VNVA SRMES TDSVGQIQVP DEVYERLKDD FVLRERGHIN VKGKGVMRTW YLIGRKVAAD PGEVRGAEPR TAGVAAA

UniProtKB: Adenylate cyclase

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Macromolecule #2: Nanobody Nb4

MacromoleculeName: Nanobody Nb4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.771179 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAQWQLVESG GGLVQAGGSL RLSCTASGII LSINSMGWYR QTAGNEREWV AFSTAGGSTT YADSVKGRFT ISRDNAKNTV YLQMNSLKP EDTAVYYCNT PAGRVGGTWG QGTPVTVSSH HHHHHEPEA

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Macromolecule #3: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: 3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ONM
Molecular weightTheoretical: 656.328 Da
Chemical component information

ChemComp-ONM:
3'-O-(N-METHYLANTHRANILOYL)-GUANOSINE-5'-TRIPHOSPHATE

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Macromolecule #4: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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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

BufferpH: 7.5 / Details: 50 mM Tris pH 7.5, 200 mM NaCl, 0.1 % digitonin
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 646042
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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