EMDB-10987: Isolated heme A synthase from Aquifex aeolicus is a trimer

Basic information

• Entry: Database: EMDB / ID: EMD-10987
• Title: Isolated heme A synthase from Aquifex aeolicus is a trimer

Sample

• Complex: Heme A synthase trimer
  • Protein or peptide: Heme A Synthase

• Biological species: Aquifex aeolicus VF5 (bacteria)
• Method: single particle reconstruction / cryo EM / Resolution: 4.2 Å
• Authors: Hui Z / Guoliang Z / Shuangbo Z / Xinmei L / Janosch M / Nina M / Fei S / Guohong P / Hao X / Hartmut M
• Citation: Journal: mBio / Year: 2020; Title: Isolated Heme A Synthase from Aquifex aeolicus Is a Trimer.; Authors: Hui Zeng / Guoliang Zhu / Shuangbo Zhang / Xinmei Li / Janosch Martin / Nina Morgner / Fei Sun / Guohong Peng / Hao Xie / Hartmut Michel / ; Abstract: The integral membrane protein heme A synthase (HAS) catalyzes the biosynthesis of heme A, which is a prerequisite for cellular respiration in a wide range of aerobic organisms. Previous studies have revealed that HAS can form homo-oligomeric complexes, and this oligomerization appears to be evolutionarily conserved among prokaryotes and eukaryotes and is shown to be essential for the biological function of eukaryotic HAS. Despite its importance, little is known about the detailed structural properties of HAS oligomers. Here, we aimed to address this critical issue by analyzing the oligomeric state of HAS from (AaHAS) using a combination of techniques, including size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), cross-linking, laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), and single-particle electron cryomicroscopy (cryo-EM). Our results show that HAS forms a thermostable trimeric complex. A cryo-EM density map provides information on the oligomerization interface of the AaHAS trimer. These results provide structural insights into HAS multimerization and expand our knowledge of this important enzyme. Heme A is a vital redox cofactor unique for the terminal cytochrome oxidase in mitochondria and many microorganisms. It plays a key role in oxygen reduction by serving as an electron carrier and as the oxygen-binding site. Heme A is synthesized from heme O by an integral membrane protein, heme A synthase (HAS). Defects in HAS impair cellular respiration and have been linked to various human diseases, e.g., fatal infantile hypertrophic cardiomyopathy and Leigh syndrome. HAS exists as a stable oligomeric complex, and studies have shown that oligomerization of eukaryotic HAS is necessary for its proper function. However, the molecular architecture of the HAS oligomeric complex has remained uncharacterized. The present study shows that HAS forms trimers and reveals how the oligomeric arrangement contributes to the complex stability and flexibility, enabling HAS to perform its catalytic function effectively. This work provides the basic understanding for future studies on heme A biosynthesis.

History

Deposition	Apr 30, 2020	-
Header (metadata) release	Jun 17, 2020	-
Map release	Jun 17, 2020	-
Update	Jun 17, 2020	-
Current status	Jun 17, 2020	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_10987.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 0.82 Å

Density

Contour Level	By AUTHOR: 0.009 / Movie #1: 0.017
Minimum - Maximum	-0.062946446 - 0.085685804
Average (Standard dev.)	0.0002741156 (±0.002229034)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 209.92 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	0.82	0.82	0.82
M x/y/z	256	256	256
origin x/y/z	0.000	0.000	0.000
length x/y/z	209.920	209.920	209.920
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	256	256	256
D min/max/mean	-0.063	0.086	0.000

Supplemental data

Sample components

Entire : Heme A synthase trimer

• Entire: Name: Heme A synthase trimer

Components

• Complex: Heme A synthase trimer
  • Protein or peptide: Heme A Synthase

Supramolecule #1: Heme A synthase trimer

• Supramolecule: Name: Heme A synthase trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
• Source (natural): Organism: Aquifex aeolicus VF5 (bacteria)
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)

Macromolecule #1: Heme A Synthase

• Macromolecule: Name: Heme A Synthase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO; EC number: Oxidoreductases; Acting on the CH-CH group of donors

Sequence

String:

MGMNTNLKSS PLKTLVLASV VLTYVLMVFG GIVTSTGSGL GCPDWPLCHG QLLPFQLKEQ IPTPPAPVVA PTPLQPWIEQ THRILGGITG IVLLATLFYA FKRGTSFVKK ALVFIFIALI LEALLGMRVV ITEAPLLREL LHYVYTSAHL ILSVFILSTI TITYYYVKFF GERPKEYIPY ADALYVATMF QILLGIFVRY VKALEYNQFV YYLHITYAGF LVILSLFIMF KEFNKYSLIT FLLMTAQILA GVATVISGFF LPYLFLHIAI GFFIVLWVSY LVAPSVLKTY TEFRGELARN SSAWSHPQFE K

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 3.5 mg/mL

Buffer

pH: 7.4
Component:

Concentration	Formula	Name
150.0 mM	NaCl	sodium chloride
20.0 mM	Tris-HCl	Tris hydrochloride

• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 %

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 52065
• Initial angle assignment: Type: COMMON LINE
• Final angle assignment: Type: MAXIMUM LIKELIHOOD