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- EMDB-25805: Cryo-EM structure of methane monooxygenase hydroxylase (by graphene) -
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Open data
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Basic information
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Title | Cryo-EM structure of methane monooxygenase hydroxylase (by graphene) | ||||||||||||
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![]() | methane monooxygenase / hydroxylase / cryo-EM / electron transport / OXIDOREDUCTASE | ||||||||||||
Function / homology | ![]() methane metabolic process / methane monooxygenase (soluble) / : / : / : / one-carbon metabolic process / metal ion binding Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.4 Å | ||||||||||||
![]() | Cho US / Kim BC | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 97.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14 KB 14 KB | Display Display | ![]() |
Images | ![]() | 110.8 KB | ||
Filedesc metadata | ![]() | 5.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7tc8MC ![]() 7tc7C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.059 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : Soluble Methane Monooxygenase Hydroxylase
Entire | Name: Soluble Methane Monooxygenase Hydroxylase |
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Components |
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-Supramolecule #1: Soluble Methane Monooxygenase Hydroxylase
Supramolecule | Name: Soluble Methane Monooxygenase Hydroxylase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: Methane monooxygenase component A alpha chain
Macromolecule | Name: Methane monooxygenase component A alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: methane monooxygenase (soluble) |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 60.717145 KDa |
Sequence | String: 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 UniProtKB: Methane monooxygenase component A alpha chain |
-Macromolecule #2: Methane monooxygenase component A beta chain
Macromolecule | Name: Methane monooxygenase component A beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: methane monooxygenase (soluble) |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 45.18466 KDa |
Sequence | String: 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 UniProtKB: Methane monooxygenase component A beta chain |
-Macromolecule #3: Methane monooxygenase component A gamma chain
Macromolecule | Name: Methane monooxygenase component A gamma chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: methane monooxygenase (soluble) |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 19.879732 KDa |
Sequence | String: MAKLGIHSND TRDAWVNKIA QLNTLEKAAE MLKQFRMDHT TPFRNSYELD NDYLWIEAKL EEKVAVLKAR AFNEVDFRHK TAFGEDAKS VLDGTVAKMN AAKDKWEAEK IHIGFRQAYK PPIMPVNYFL DGERQLGTRL MELRNLNYYD TPLEELRKQR G VRVVHLQS PH UniProtKB: Methane monooxygenase component A gamma chain |
-Macromolecule #4: FE (III) ION
Macromolecule | Name: FE (III) ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: FE |
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Molecular weight | Theoretical: 55.845 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7 / Details: 30 mM HEPES, pH 7, 150 mM NaCl, 1 mM TCEP |
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 325000 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |