Pseudomonas fluorescens G150T isocyanide hydratase at 298 K XFEL data, thioimidate intermediate

Experimental Data Snapshot

  • Resolution: 1.30 Å
  • R-Value Free: 0.171 
  • R-Value Work: 0.137 
  • R-Value Observed: 0.138 

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Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography.

Smith, N.Dasgupta, M.Wych, D.C.Dolamore, C.Sierra, R.G.Lisova, S.Marchany-Rivera, D.Cohen, A.E.Boutet, S.Hunter, M.S.Kupitz, C.Poitevin, F.Moss 3rd, F.R.Mittan-Moreau, D.W.Brewster, A.S.Sauter, N.K.Young, I.D.Wolff, A.M.Tiwari, V.K.Kumar, N.Berkowitz, D.B.Hadt, R.G.Thompson, M.C.Follmer, A.H.Wall, M.E.Wilson, M.A.

(2024) Sci Adv 10: eadk7201-eadk7201

  • DOI: https://doi.org/10.1126/sciadv.adk7201
  • Primary Citation of Related Structures:  
    8VPW, 8VQ1

  • PubMed Abstract: 

    Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp 17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

  • Organizational Affiliation

    Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.

Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Isonitrile hydratase InhA231Pseudomonas fluorescensMutation(s): 1 
Gene Names: inhAPFL_4109
Find proteins for Q4K977 (Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5))
Explore Q4K977 
Go to UniProtKB:  Q4K977
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ4K977
Sequence Annotations
  • Reference Sequence
Small Molecules
Experimental Data & Validation

Experimental Data

  • Resolution: 1.30 Å
  • R-Value Free: 0.171 
  • R-Value Work: 0.137 
  • R-Value Observed: 0.138 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 72.136α = 90
b = 59.758β = 115.883
c = 56.11γ = 90
Software Package:
Software NamePurpose
cctbx.xfeldata reduction
cctbx.xfel.mergedata scaling

Structure Validation

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Ligand Structure Quality Assessment 

Entry History & Funding Information

Deposition Data

Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)United StatesR01GM139978

Revision History  (Full details and data files)

  • Version 1.0: 2024-02-21
    Type: Initial release
  • Version 1.1: 2024-04-10
    Changes: Database references