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- EMDB-17963: Structure of AHIR determined by cryoEM at 100 keV -

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Basic information

Entry
Database: EMDB / ID: EMD-17963
TitleStructure of AHIR determined by cryoEM at 100 keV
Map data
Sample
  • Complex: Acetohydroxy acid isomeroreductase
    • Protein or peptide: Ketol-acid reductoisomerase (NADP(+))
KeywordsOXIDOREDUCTASE
Function / homology
Function and homology information


ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / L-valine biosynthetic process / isoleucine biosynthetic process / NADP binding / magnesium ion binding / cytosol
Similarity search - Function
Ketol-acid reductoisomerase, prokaryotic / Ketol-acid reductoisomerase, C-terminal / Ketol-acid reductoisomerase / Ketol-acid reductoisomerase, N-terminal / Ketol-acid reductoisomerase, C-terminal domain / Acetohydroxy acid isomeroreductase, NADPH-binding domain / KARI N-terminal domain profile. / KARI C-terminal domain profile. / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Ketol-acid reductoisomerase (NADP(+))
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMcMullan G / Naydenova K / Mihaylov D / Peet MJ / Wilson H / Yamashita K / Dickerson JL / Chen S / Cannone G / Lee Y ...McMullan G / Naydenova K / Mihaylov D / Peet MJ / Wilson H / Yamashita K / Dickerson JL / Chen S / Cannone G / Lee Y / Hutchings KA / Gittins O / Sobhy M / Wells T / El-Gomati MM / Dalby J / Meffert M / Schulze-Briese C / Henderson R / Russo CJ
Funding support United Kingdom, 6 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC UP 120117 United Kingdom
Medical Research Council (MRC, United Kingdom)MC U105184322 United Kingdom
Wellcome Trust220526/B/20/Z United Kingdom
Engineering and Physical Sciences Research CouncilR122522 United Kingdom
Innovate UK103806 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T003677/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structure determination by cryoEM at 100 keV.
Authors: Greg McMullan / Katerina Naydenova / Daniel Mihaylov / Keitaro Yamashita / Mathew J Peet / Hugh Wilson / Joshua L Dickerson / Shaoxia Chen / Giuseppe Cannone / Yang Lee / Katherine A ...Authors: Greg McMullan / Katerina Naydenova / Daniel Mihaylov / Keitaro Yamashita / Mathew J Peet / Hugh Wilson / Joshua L Dickerson / Shaoxia Chen / Giuseppe Cannone / Yang Lee / Katherine A Hutchings / Olivia Gittins / Mohamed A Sobhy / Torquil Wells / Mohamed M El-Gomati / Jason Dalby / Matthias Meffert / Clemens Schulze-Briese / Richard Henderson / Christopher J Russo /
Abstract: Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose- ...Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.
History
DepositionJul 17, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateDec 6, 2023-
Current statusDec 6, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17963.png

Downloads & links

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Map

FileDownload / File: emd_17963.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 420 pix.
= 349.23 Å		0.83 Å/pix.
x 420 pix.
= 349.23 Å		0.83 Å/pix.
x 420 pix.
= 349.23 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8315 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.074770324 - 0.13057293
Average (Standard dev.)0.00016161906 (±0.0031434887)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 349.23 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17963_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

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Half map: #1

Fileemd_17963_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17963_half_map_2.map
Projections & Slices
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Sample components

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Entire : Acetohydroxy acid isomeroreductase

EntireName: Acetohydroxy acid isomeroreductase
Components
  • Complex: Acetohydroxy acid isomeroreductase
    • Protein or peptide: Ketol-acid reductoisomerase (NADP(+))

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Supramolecule #1: Acetohydroxy acid isomeroreductase

SupramoleculeName: Acetohydroxy acid isomeroreductase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermus thermophilus (bacteria)

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Macromolecule #1: Ketol-acid reductoisomerase (NADP(+))

MacromoleculeName: Ketol-acid reductoisomerase (NADP(+)) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ketol-acid reductoisomerase (NADP+)
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 37.994246 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTGTMKIYYE HDADLGFILG KKVAVLGFGS QGHAHAHNLK DSGVDVRVGL RKGSRSWEKA EAAGLRVLPV AEAVREADVV MVLLPDEKQ AQVYREEVEP NLKEGGALAF AHGFNVHFGQ IKPRKDLDVW MVAPKGPGHL VRSEYGGGSN WSHPQFEKRP P GGSGVPAL ...String:
MTGTMKIYYE HDADLGFILG KKVAVLGFGS QGHAHAHNLK DSGVDVRVGL RKGSRSWEKA EAAGLRVLPV AEAVREADVV MVLLPDEKQ AQVYREEVEP NLKEGGALAF AHGFNVHFGQ IKPRKDLDVW MVAPKGPGHL VRSEYGGGSN WSHPQFEKRP P GGSGVPAL VAVHQDASGS AFPTALAYAK AIGAARAGVI ATTFKDETET DLFGEQAVLC GGLTRLIRAG FETLVEAGYP PE MAYFETV HEVKLIVDLI YEAGLKGMRY SISNTAEYGD YTRGDLAVPL EETKRRMREI LRQIQSGEFA REWMLENQVG SPV LEANRK RWAAHPIEEV GSRLRAMMRS

UniProtKB: Ketol-acid reductoisomerase (NADP(+))

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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: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL 1400/HR + YPS FEG
Image recordingFilm or detector model: DECTRIS SINGLA (1k x 1k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

13256

Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 15220
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL
Output model

PDB-8pve:
Structure of AHIR determined by cryoEM at 100 keV

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