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- PDB-8c8l: In vitro structure of the Nitrosopumilus maritimus S-layer - Two-... -

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Basic information

Entry
Database: PDB / ID: 8c8l
TitleIn vitro structure of the Nitrosopumilus maritimus S-layer - Two-fold symmetry (C2)
ComponentsCell surface protein
KeywordsSTRUCTURAL PROTEIN / Nmar_1547 S-layer
Function / homologymembrane / Uncharacterized protein
Function and homology information
Biological speciesNitrosopumilus maritimus SCM1 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 Å
Authorsvon Kuegelgen, A. / Bharat, T.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/31 United Kingdom
CitationJournal: Nature / Year: 2024
Title: Membraneless channels sieve cations in ammonia-oxidizing marine archaea.
Authors: Andriko von Kügelgen / C Keith Cassidy / Sofie van Dorst / Lennart L Pagani / Christopher Batters / Zephyr Ford / Jan Löwe / Vikram Alva / Phillip J Stansfeld / Tanmay A M Bharat /
Abstract: Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine ...Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.
History
DepositionJan 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jun 12, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jun 19, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cell surface protein
B: Cell surface protein
C: Cell surface protein
D: Cell surface protein
E: Cell surface protein
F: Cell surface protein


Theoretical massNumber of molelcules
Total (without water)1,098,9366
Polymers1,098,9366
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14A
24E
15A
25F
16A
26B
17A
27C
18A
28D
19A
29E
110A
210F
111B
211C
112B
212D
113B
213E
114B
214F
115B
215C
116B
216D
117B
217E
118B
218F
119C
219D
120C
220E
121C
221F
122C
222D
123C
223E
124C
224F
125D
225E
126D
226F
127D
227E
128D
228F
129E
229F
130E
230F

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A37 - 1616
2010B37 - 1616
1020A37 - 1616
2020C37 - 1616
1030A37 - 1616
2030D37 - 1616
1040A37 - 1616
2040E37 - 1616
1050A37 - 1616
2050F37 - 1616
1060A2153 - 2163
2060B2153 - 2163
1070A2153 - 2163
2070C2153 - 2163
1080A2153 - 2163
2080D2153 - 2163
1090A2153 - 2163
2090E2153 - 2163
10100A2153 - 2163
20100F2153 - 2163
10110B37 - 1616
20110C37 - 1616
10120B37 - 1616
20120D37 - 1616
10130B37 - 1616
20130E37 - 1616
10140B37 - 1616
20140F37 - 1616
10150B2153 - 2163
20150C2153 - 2163
10160B2153 - 2163
20160D2153 - 2163
10170B2153 - 2163
20170E2153 - 2163
10180B2153 - 2163
20180F2153 - 2163
10190C37 - 1616
20190D37 - 1616
10200C37 - 1616
20200E37 - 1616
10210C37 - 1616
20210F37 - 1616
10220C2153 - 2163
20220D2153 - 2163
10230C2153 - 2163
20230E2153 - 2163
10240C2153 - 2163
20240F2153 - 2163
10250D37 - 1616
20250E37 - 1616
10260D37 - 1616
20260F37 - 1616
10270D2153 - 2163
20270E2153 - 2163
10280D2153 - 2163
20280F2153 - 2163
10290E37 - 1616
20290F37 - 1616
10300E2153 - 2163
20300F2153 - 2163

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

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Components

#1: Protein
Cell surface protein


Mass: 183156.000 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Details: In-vitro isolated S-layer / Source: (natural) Nitrosopumilus maritimus SCM1 (archaea) / Strain: SCM1 / References: UniProt: A9A4Y9
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Nitrosopumilus maritimus S-layer / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Nitrosopumilus maritimus S-layer C2 symmetrised / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Nitrosopumilus maritimus SCM1 (archaea) / Strain: SCM1 / Cellular location: extracellular
Buffer solutionpH: 7.5
Details: 50 mM HEPES/NaOH pH=7.5, 500 mM NaCl, 50 mM MgCl2, 10 mM CaCl2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2500 mMNaClNaCl1
350 mMMgCl2MgCl21
410 mMCaCl2CaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: In vitro isolate S-layer cell envelopes
Specimen supportDetails: 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 283.15 K
Details: absorption for 60 sec and blotted for 5 sec with blot force -10

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 81000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 4.2 sec. / Electron dose: 48.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 12557
Details: collected over three sessions with one session with the stage tilted by 33 degrees (alpha tilt)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Chromatic aberration corrector: not used / Energyfilter slit width: 20 eV / Spherical aberration corrector: not used
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Topaz0.2.5particle selectionResNet8 trained model
2RELIONparticle selection
3EPUimage acquisition
5CTFFIND4.1.13CTF correctionCTFFIND4 was used as implemented in RELION 3.1
8Coot0.9.2-premodel fitting
10PHENIX1.19-4092model refinement
11REFMACmodel refinement
12RELION3.1initial Euler assignment
13RELION3.1final Euler assignment
14RELION3.1classification
15RELION3.13D reconstruction
Image processingDetails: Movies collected at the scope were clustered into optics groups based on the XML meta-data of the data-collection software EPU (Thermo Fisher Scientific) using a k-means algorithm ...Details: Movies collected at the scope were clustered into optics groups based on the XML meta-data of the data-collection software EPU (Thermo Fisher Scientific) using a k-means algorithm implemented in EPU_group_AFIS (https://github.com/DustinMorado/EPU_group_AFIS). Imported movies were motion-corrected, dose-weighted, and Fourier cropped (2x) with MotionCor2 implemented in RELION-3.1. Contrast transfer functions (CTFs) of the resulting motion-corrected micrographs were estimated using CTFFIND4.
CTF correctionDetails: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1971908
Details: Initially, side views of S-layer sheets were first manually picked along the edge of the lattice using the helical picking tab in RELION while setting the helical rise to 60 angstrom. Top ...Details: Initially, side views of S-layer sheets were first manually picked along the edge of the lattice using the helical picking tab in RELION while setting the helical rise to 60 angstrom. Top and tilted views were manually picked at the central hexameric axis. Manually picked particles were extracted in 4x downsampled 128x128 pixel2 boxes and classified using reference-free 2D classification inside RELION-3.1. Class averages centered at a hexameric axis were used to automatically pick particles inside RELION-3.1. Automatically picked particles were extracted in 4x downsampled 128x128 pixel2 boxes and classified using reference-free 2D classification. Particle coordinates belonging to class averages centered at the hexameric axis were used to train TOPAZ in 5x downsampled micrographs with the neural network architecture conv127. For the final reconstruction, particles were picked using TOPAZ and the previously trained neural network above. Additionally, top, bottom, and side views were picked using the reference-based autopicker inside RELION-3.1, which TOPAZ did not readily identify. Particles were extracted in 4x downsampled 128x128 pixel2 boxes and classified using reference-free 2D classification inside RELION-3.1. Particles belonging to class averages centered at the hexameric axis were combined, and particles within 30 angstrom were removed to prevent duplication after alignment. All resulting particles were then re-extracted in 4x downsampled 128x128 pixel2 boxes.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 354860 / Algorithm: FOURIER SPACE
Details: Per-particle defocus, anisotropy magnification, and higher-order aberrations were refined inside RELION3.1, followed by three rounds of focused 3D auto refinement. Bayesian particle ...Details: Per-particle defocus, anisotropy magnification, and higher-order aberrations were refined inside RELION3.1, followed by three rounds of focused 3D auto refinement. Bayesian particle polishing was performed subsequently in a 640x640 pixel2 box followed by auto-refinement and symmetry relaxation. The final map was obtained from 354,860 particles and post-processed using a soft mask focused on the central hexamer, yielding a global resolution of 2.7 angstrom according to the Fourier shell correlation criterion between two independently refined half-maps at a threshold value at 0.143, and local resolution up to 2.5 angstrom
Symmetry type: POINT
Atomic model buildingB value: 41.4 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Best Fit
RefinementResolution: 2.71→2.71 Å / Cor.coef. Fo:Fc: 0.909 / SU B: 12.215 / SU ML: 0.232 / ESU R: 0.32
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.32231 --
obs0.32231 830747 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 76.059 Å2
Baniso -1Baniso -2Baniso -3
1--3.45 Å20.84 Å2-0 Å2
2---3.88 Å20 Å2
3---7.32 Å2
Refinement stepCycle: 1 / Total: 72246
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0120.01374446
ELECTRON MICROSCOPYr_bond_other_d0.0010.01764473
ELECTRON MICROSCOPYr_angle_refined_deg1.4751.669100326
ELECTRON MICROSCOPYr_angle_other_deg1.4851.606149157
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.21359474
ELECTRON MICROSCOPYr_dihedral_angle_2_deg34.725.5963624
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.9521510578
ELECTRON MICROSCOPYr_dihedral_angle_4_deg22.08915210
ELECTRON MICROSCOPYr_chiral_restr0.0660.210776
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.0285914
ELECTRON MICROSCOPYr_gen_planes_other0.0030.0215467
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.27.87938271
ELECTRON MICROSCOPYr_mcbond_other6.27.87938269
ELECTRON MICROSCOPYr_mcangle_it9.14811.85247382
ELECTRON MICROSCOPYr_mcangle_other9.14811.85247383
ELECTRON MICROSCOPYr_scbond_it7.8698.62536175
ELECTRON MICROSCOPYr_scbond_other7.8688.62536176
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other12.22712.60752945
ELECTRON MICROSCOPYr_long_range_B_refined18.65296178
ELECTRON MICROSCOPYr_long_range_B_other18.65296179
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A963600.03
12B963600.03
21A963960.03
22C963960.03
31A969800.02
32D969800.02
41A963580.03
42E963580.03
51A962580.03
52F962580.03
61A6220.05
62B6220.05
71A6200.08
72C6200.08
81A6260.05
82D6260.05
91A6220.05
92E6220.05
101A6220.05
102F6220.05
111B964020.03
112C964020.03
121B964620.03
122D964620.03
131B970220.02
132E970220.02
141B962460.03
142F962460.03
151B6280.06
152C6280.06
161B6280.01
162D6280.01
171B6340
172E6340
181B6320.02
182F6320.02
191C965020.03
192D965020.03
201C964260.02
202E964260.02
211C969480.02
212F969480.02
221C6280.06
222D6280.06
231C6280.06
232E6280.06
241C6340.08
242F6340.08
251D964500.03
252E964500.03
261D963540.03
262F963540.03
271D6280.01
272E6280.01
281D6300.02
282F6300.02
291E962900.03
292F962900.03
301E6320.02
302F6320.02
LS refinement shellResolution: 2.72→2.791 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.75 61454 -
obs--100 %

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