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- EMDB-25805: Cryo-EM structure of methane monooxygenase hydroxylase (by graphene) -

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Basic information

Entry
Database: EMDB / ID: EMD-25805
TitleCryo-EM structure of methane monooxygenase hydroxylase (by graphene)
Map data
Sample
  • Complex: Soluble Methane Monooxygenase Hydroxylase
    • Protein or peptide: Methane monooxygenase component A alpha chain
    • Protein or peptide: Methane monooxygenase component A beta chain
    • Protein or peptide: Methane monooxygenase component A gamma chain
  • Ligand: FE (III) ION
Function / homology
Function and homology information


methane monooxygenase (soluble) / methane monooxygenase NADH activity / methane monooxygenase NADPH activity / methane metabolic process / : / one-carbon metabolic process / metal ion binding
Similarity search - Function
Methane monooxygenase, gamma chain / Methane monooxygenase, gamma chain, domain 1 / Methane monooxygenase, gamma chain, domain 2 / Methane monooxygenase, gamma chain superfamily / Methane monooxygenase, hydrolase gamma chain / Methane/phenol monooxygenase, hydroxylase component / Propane/methane/phenol/toluene hydroxylase / Methane/Phenol/Alkene Hydroxylase / Ribonucleotide reductase-like / Ferritin-like superfamily
Similarity search - Domain/homology
Methane monooxygenase component A gamma chain / Methane monooxygenase component A beta chain / Methane monooxygenase component A alpha chain
Similarity search - Component
Biological speciesMethylococcus capsulatus str. Bath (bacteria) / Methylococcus capsulatus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsCho US / Kim BC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK111465 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS116008 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA250329 United States
CitationJournal: ACS Nano / Year: 2023
Title: Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Soluble Methane Monooxygenase Hydroxylase.
Authors: Eungjin Ahn / Byungchul Kim / Soyoung Park / Amanda L Erwin / Suk Hyun Sung / Robert Hovden / Shyamal Mosalaganti / Uhn-Soo Cho /
Abstract: Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for ...Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air-water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air-water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.
History
DepositionDec 23, 2021-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25805.png

Downloads & links

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Map

FileDownload / File: emd_25805.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 1.25
Minimum - Maximum-6.9823675 - 10.849868
Average (Standard dev.)-0.0016008232 (±0.26426223)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Soluble Methane Monooxygenase Hydroxylase

EntireName: Soluble Methane Monooxygenase Hydroxylase
Components
  • Complex: Soluble Methane Monooxygenase Hydroxylase
    • Protein or peptide: Methane monooxygenase component A alpha chain
    • Protein or peptide: Methane monooxygenase component A beta chain
    • Protein or peptide: Methane monooxygenase component A gamma chain
  • Ligand: FE (III) ION

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Supramolecule #1: Soluble Methane Monooxygenase Hydroxylase

SupramoleculeName: Soluble Methane Monooxygenase Hydroxylase / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Methylococcus capsulatus str. Bath (bacteria)

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Macromolecule #1: Methane monooxygenase component A alpha chain

MacromoleculeName: Methane monooxygenase component A alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: methane monooxygenase (soluble)
Source (natural)Organism: Methylococcus capsulatus (bacteria) / Strain: ATCC 33009 / NCIMB 11132 / Bath
Molecular weightTheoretical: 60.717145 KDa
SequenceString: MALSTATKAA TDALAANRAP TSVNAQEVHR WLQSFNWDFK NNRTKYATKY KMANETKEQF KLIAKEYARM EAVKDERQFG SLQDALTRL NAGVRVHPKW NETMKVVSNF LEVGEYNAIA ATGMLWDSAQ AAEQKNGYLA QVLDEIRHTH QCAYVNYYFA K NGQDPAGH ...String:
MALSTATKAA TDALAANRAP TSVNAQEVHR WLQSFNWDFK NNRTKYATKY KMANETKEQF KLIAKEYARM EAVKDERQFG SLQDALTRL NAGVRVHPKW NETMKVVSNF LEVGEYNAIA ATGMLWDSAQ AAEQKNGYLA QVLDEIRHTH QCAYVNYYFA K NGQDPAGH NDARRTRTIG PLWKGMKRVF SDGFISGDAV ECSLNLQLVG EACFTNPLIV AVTEWAAANG DEITPTVFLS IE TDELRHM ANGYQTVVSI ANDPASAKYL NTDLNNAFWT QQKYFTPVLG MLFEYGSKFK VEPWVKTWNR WVYEDWGGIW IGR LGKYGV ESPRSLKDAK QDAYWAHHDL YLLAYALWPT GFFRLALPDQ EEMEWFEANY PGWYDHYGKI YEEWRARGCE DPSS GFIPL MWFIENNHPI YIDRVSQVPF CPSLAKGAST LRVHEYNGQM HTFSDQWGER MWLAEPERYE CQNIFEQYEG RELSE VIAE LHGLRSDGKT LIAQPHVRGD KLWTLDDIKR LNCVFKNPVK AFN

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Macromolecule #2: Methane monooxygenase component A beta chain

MacromoleculeName: Methane monooxygenase component A beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: methane monooxygenase (soluble)
Source (natural)Organism: Methylococcus capsulatus (bacteria)
Molecular weightTheoretical: 45.18466 KDa
SequenceString: MSMLGERRRG LTDPEMAAVI LKALPEAPLD GNNKMGYFVT PRWKRLTEYE ALTVYAQPNA DWIAGGLDWG DWTQKFHGGR PSWGNETTE LRTVDWFKHR DPLRRWHAPY VKDKAEEWRY TDRFLQGYSA DGQIRAMNPT WRDEFINRYW GAFLFNEYGL F NAHSQGAR ...String:
MSMLGERRRG LTDPEMAAVI LKALPEAPLD GNNKMGYFVT PRWKRLTEYE ALTVYAQPNA DWIAGGLDWG DWTQKFHGGR PSWGNETTE LRTVDWFKHR DPLRRWHAPY VKDKAEEWRY TDRFLQGYSA DGQIRAMNPT WRDEFINRYW GAFLFNEYGL F NAHSQGAR EALSDVTRVS LAFWGFDKID IAQMIQLERG FLAKIVPGFD ESTAVPKAEW TNGEVYKSAR LAVEGLWQEV FD WNESAFS VHAVYDALFG QFVRREFFQR LAPRFGDNLT PFFINQAQTY FQIAKQGVQD LYYNCLGDDP EFSDYNRTVM RNW TGKWLE PTIAALRDFM GLFAKLPAGT TDKEEITASL YRVVDDWIED YASRIDFKAD RDQIVKAVLA GLK

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Macromolecule #3: Methane monooxygenase component A gamma chain

MacromoleculeName: Methane monooxygenase component A gamma chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: methane monooxygenase (soluble)
Source (natural)Organism: Methylococcus capsulatus (bacteria) / Strain: ATCC 33009 / NCIMB 11132 / Bath
Molecular weightTheoretical: 19.879732 KDa
SequenceString:
MAKLGIHSND TRDAWVNKIA QLNTLEKAAE MLKQFRMDHT TPFRNSYELD NDYLWIEAKL EEKVAVLKAR AFNEVDFRHK TAFGEDAKS VLDGTVAKMN AAKDKWEAEK IHIGFRQAYK PPIMPVNYFL DGERQLGTRL MELRNLNYYD TPLEELRKQR G VRVVHLQS PH

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Macromolecule #4: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: FE
Molecular weightTheoretical: 55.845 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 / Details: 30 mM HEPES, pH 7, 150 mM NaCl, 1 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1mty

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 325000

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