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- EMDB-38355: Structure of the Argonaute protein from Kurthia massiliensis in c... -

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Entry
Database: EMDB / ID: EMD-38355
TitleStructure of the Argonaute protein from Kurthia massiliensis in complex with guide DNA
Map data
Sample
  • Complex: Structure of the Argonaute protein from Kurthia massiliensis in complex with guide DNA
    • Protein or peptide: KmAgo
    • Other: guide DNA
  • Ligand: MANGANESE (II) ION
  • Ligand: water
KeywordsMesophilic prokaryotic Argonaute / DNA BINDING PROTEIN-DNA COMPLEX
Biological speciesKurthia massiliensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsXin T / Hui D / Shaowen W
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2021YFC2100100 China
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structural and mechanistic insights into a mesophilic prokaryotic Argonaute.
Authors: Xin Tao / Hui Ding / Shaowen Wu / Fei Wang / Hu Xu / Jie Li / Chao Zhai / Shunshun Li / Kai Chen / Shan Wu / Yang Liu / Lixin Ma /
Abstract: Argonaute (Ago) proteins are programmable nucleases found in all domains of life, playing a crucial role in biological processes like DNA/RNA interference and gene regulation. Mesophilic prokaryotic ...Argonaute (Ago) proteins are programmable nucleases found in all domains of life, playing a crucial role in biological processes like DNA/RNA interference and gene regulation. Mesophilic prokaryotic Agos (pAgos) have gained increasing research interest due to their broad range of potential applications, yet their molecular mechanisms remain poorly understood. Here, we present seven cryo-electron microscopy structures of Kurthia massiliensis Ago (KmAgo) in various states. These structures encompass the steps of apo-form, guide binding, target recognition, cleavage, and release, revealing that KmAgo employs a unique DDD catalytic triad, instead of a DEDD tetrad, for DNA target cleavage under 5'P-DNA guide conditions. Notably, the last catalytic residue, D713, is positioned outside the catalytic pocket in the absence of guide. After guide binding, D713 enters the catalytic pocket. In contrast, the corresponding catalytic residue in other Agos has been consistently located in the catalytic pocket. Moreover, we identified several sites exhibiting enhanced catalytic activity through alanine mutagenesis. These sites have the potential to serve as engineering targets for augmenting the catalytic efficiency of KmAgo. This structural analysis of KmAgo advances the understanding of the diversity of molecular mechanisms by Agos, offering insights for developing and optimizing mesophilic pAgos-based programmable DNA and RNA manipulation tools.
History
DepositionDec 18, 2023-
Header (metadata) releaseJan 1, 2025-
Map releaseJan 1, 2025-
UpdateJan 8, 2025-
Current statusJan 8, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38355.png

Downloads & links

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Map

FileDownload / File: emd_38355.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.856 Å		0.85 Å/pix.
x 256 pix.
= 217.856 Å		0.85 Å/pix.
x 256 pix.
= 217.856 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.851 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.1664877 - 4.380801
Average (Standard dev.)-0.0005436435 (±0.081048794)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.856 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_38355_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_38355_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of the Argonaute protein from Kurthia massiliensis in c...

EntireName: Structure of the Argonaute protein from Kurthia massiliensis in complex with guide DNA
Components
  • Complex: Structure of the Argonaute protein from Kurthia massiliensis in complex with guide DNA
    • Protein or peptide: KmAgo
    • Other: guide DNA
  • Ligand: MANGANESE (II) ION
  • Ligand: water

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Supramolecule #1: Structure of the Argonaute protein from Kurthia massiliensis in c...

SupramoleculeName: Structure of the Argonaute protein from Kurthia massiliensis in complex with guide DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Kurthia massiliensis (bacteria)

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Macromolecule #1: KmAgo

MacromoleculeName: KmAgo / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Kurthia massiliensis (bacteria)
Molecular weightTheoretical: 85.485203 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEAYITEMVS RERANELEVY VYVFPRKQSD NNYEGVYHIM RAWQRANDLP LAYNQHTIMA FSPVRHMCGY TPMETQKRHI NIDSPFERA LLERLIKNSL IFTAERHLHA KRVGHALRLN QVQQIRQVII YEAIELYVNI IENRISIGFH LTHQFEYVYT L QSMIEQGK ...String:
MEAYITEMVS RERANELEVY VYVFPRKQSD NNYEGVYHIM RAWQRANDLP LAYNQHTIMA FSPVRHMCGY TPMETQKRHI NIDSPFERA LLERLIKNSL IFTAERHLHA KRVGHALRLN QVQQIRQVII YEAIELYVNI IENRISIGFH LTHQFEYVYT L QSMIEQGK TIRPGMRVVH SNGRQHYTYT VENVATYGVT DRCPLLQTSI YQYYVEKGAQ HILRTFTRST RVIHVRTKEQ RL SYAATLL KPLCTFETMQ PQDVLNVSKC IKLSASKRMK CTYRWIQQLR AQYRHLTFAP NPFTIAQNGY KLDQLSTPKV HFH RDYATV VSGMKTGKLY KGGNIKISVL FDEDFYLKHH ITKKDIYQFI AVLQKIAIAQ GVNMTISTST KSITGKFTDD FFHH FTEEV EALQPIFAQT TVLAFITSTH LSNKKTRSYQ LLKQYFGGKW DIASQVITEK TIEAFQKILH KHGLKNFYPN DEQHC LRVI DVLKNESFYY TVMNILLGVY VKSGIQPWIL ANTTHSDCFI GIDVSHENGN SAAGMMNVIG SQGHLIQQAP LNGILA GEK IDDTLLANLL KQMIKAYHTQ FQRFPKHITI HRDGFWREHT ALVEKIMSHY EITYDIVEII KKPNRRMAFF NSVDNTF ST RQGTVYQRGN EAFLCATNPQ QKVGMAQPIK IHQVTKTLPF SHIIEDVYNL SFLHIHAMNK MRLPATIHYA DLSATAYQ R GQVMPRSGNQ TNLPFV

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Macromolecule #2: guide DNA

MacromoleculeName: guide DNA / type: other / ID: 2 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Kurthia massiliensis (bacteria)
Molecular weightTheoretical: 5.817656 KDa
SequenceString:
(DT)GAGG(DT)AG(DT)A GG(DT)(DT)G(DT)A(DT)

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Macromolecule #3: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 24 / 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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 381463
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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