PDBsum entry 2uuk

Hydrolase/hydrolase inhibitor

PDB id: 2uuk

Contents

Protein chains

28 a.a. *

252 a.a. *

Ligands

ASP-PHE-GLU-GLU-ILE-PRO-GLU-GLU-TYS-LEU

897

Metals

_NA

_CA

Waters ×345

* Residue conservation analysis

PDB id:

2uuk

Name:

Hydrolase/hydrolase inhibitor

Title:

Thrombin-hirugen-gw420128 ternary complex at 1.39a resolution

Structure:

Human alpha thrombin. Chain: a. Fragment: residues 328-363. Thrombin. Chain: b. Fragment: residues 364-622. Synonym: human alpha thrombin, coagulation factor ii. Hirudin i. Chain: h.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Tissue: blood plasma. Synthetic: yes. Hirudo medicinalis. Medicinal leech. Organism_taxid: 6421. Other_details: this polypeptide chain was chemically synthesized.

Resolution:

1.39Å R-factor: 0.118 R-free: 0.173

Authors:

H.U.Ahmed,M.P.Blakeley,M.Cianci,D.W.J.Cruickshank,J.A.Hubbard, J.R.Helliwell

Key ref:

H.U.Ahmed et al. (2007). The determination of protonation states in proteins. Acta Crystallogr D Biol Crystallogr, 63, 906-922. PubMed id: 17642517 DOI: 10.1107/S0907444907029976

Date:

03-Mar-07 Release date: 04-Sep-07

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 28 a.a.

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 252 a.a.

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.4.21.5 - thrombin.
• Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.

DOI no: 10.1107/S0907444907029976

Acta Crystallogr D Biol Crystallogr 63:906-922 (2007)

PubMed id: 17642517

The determination of protonation states in proteins.

H.U.Ahmed, M.P.Blakeley, M.Cianci, D.W.Cruickshank, J.A.Hubbard, J.R.Helliwell.

ABSTRACT

The protonation states of aspartic acids and glutamic acids as well as histidine are investigated in four X-ray cases: Ni,Ca concanavalin A at 0.94 A, a thrombin-hirugen binary complex at 1.26 A resolution and two thrombin-hirugen-inhibitor ternary complexes at 1.32 and 1.39 A resolution. The truncation of the Ni,Ca concanavalin A data at various test resolutions between 0.94 and 1.50 A provided a test comparator for the ;unknown' thrombin-hirugen carboxylate bond lengths. The protonation states of aspartic acids and glutamic acids can be determined (on the basis of convincing evidence) even to the modest resolution of 1.20 A as exemplified by our X-ray crystal structure refinements of Ni and Mn concanavalin A and also as indicated in the 1.26 A structure of thrombin, both of which are reported here. The protonation-state indication of an Asp or a Glu is valid provided that the following criteria are met (in order of importance). (i) The acidic residue must have a single occupancy. (ii) Anisotropic refinement at a minimum diffraction resolution of 1.20 A (X-ray data-to-parameter ratio of approximately 3.5:1) is required. (iii) Both of the bond lengths must agree with the expectation (i.e. dictionary values), thus allowing some relaxation of the bond-distance standard uncertainties required to approximately 0.025 A for a '3sigma' determination or approximately 0.04 A for a '2sigma' determination, although some variation of the expected bond-distance values must be allowed according to the microenvironment of the hydrogen of interest. (iv) Although the F(o) - F(c) map peaks are most likely to be unreliable at the resolution range around 1.20 A, if admitted as evidence the peak at the hydrogen position must be greater than or equal to 2.5 sigma and in the correct geometry. (v) The atomic B factors need to be less than 10 A(2) for bond-length differentiation; furthermore, the C=O bond can also be expected to be observed with continuous 2F(o) - F(c) electron density and the C-OH bond with discontinuous electron density provided that the atomic B factors are less than approximately 20 A(2) and the contour level is increased. The final decisive option is to carry out more than one experiment, e.g. multiple X-ray crystallography experiments and ideally neutron crystallography. The complementary technique of neutron protein crystallography has provided evidence of the protonation states of histidine and acidic residues in concanavalin A and also the correct orientations of asparagine and glutamine side chains. Again, the truncation of the neutron data at various test resolutions between 2.5 and 3.0 A, even 3.25 and 3.75 A resolution, examines the limits of the neutron probe. These various studies indicate a widening of the scope of both X-ray and neutron probes in certain circumstances to elucidate the protonation states in proteins.

Selected figure(s)

Figure 6.
Figure 6 Asp28 carboxyl-group bond lengths (restrained) versus resolution for Ni,Ca concanavalin A and native (Mn,Ca) concanavalin A refined using SHELXL-97. As expected, with restraints on the dictionary 1.249 Å bond length dominates from 1.35 to 1.50 Å resolution. This is true in both cases, but Ni,Ca concanavalin A `behaves' better.

Figure 16.
Figure 16 Positive neutron F[o] - F[c] maps for His24 (all at 2.5 ) at various resolutions (calculated from the model with no D atoms included). (a) 2.2 Å resolution; (b) 2.5 Å resolution; (c) 2.75 Å resolution; (d) 3.0 Å resolution; (e) 3.25 Å resolution; (f) 3.5 Å resolution; (g) 3.75 Å resolution.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2007, 63, 906-922) copyright 2007.

Figures were selected by the author.

Author's comment

Synopsis The scope of X-ray and neutron protein crystallography to determine protonation states of functionally important amino acids is investigated at resolutions from 0.94Å to 1.50Å and from 2.2Å to 3.75Å respectively using concanavalin A as a test case. Key indicators are then combined to assign levels of confidence of such protonation states for three thrombin X-ray crystal structures at resolutions of 1.26Å, 1.32Å and 1.39Å. These various studies indicate a widening of the scope of both X-ray and neutron probes in certain circumstances to elucidate the protonation states in proteins.