PDBsum entry 5uvl

Hydrolase

PDB id: 5uvl

Contents

Protein chain

279 a.a.

Ligands

NO3 ×2

Metals

_CA ×2

PDB id:

5uvl

Name:

Hydrolase

Title:

Serial millisecond crystallography of membrane and soluble protein micro-crystals using synchrotron radiation

Structure:

Proteinase k. Chain: a. Synonym: endopeptidase k,tritirachium alkaline proteinase. Engineered: yes

Source:

Parengyodontium album. Organism_taxid: 37998. Gene: prok. Expressed in: unidentified. Expression_system_taxid: 32644

Resolution:

2.65Å R-factor: 0.228 R-free: 0.243

Authors:

J.M.Martin-Garcia,C.E.Conrad,G.Nelson,N.Stander,N.A.Zatsepin,J.Zook, L.Zhu,J.Geiger,E.Chun,D.Kissick,M.C.Hilgart,C.Ogata,A.Ishchenko, N.Nagaratnam,S.Roy-Chowdhury,J.Coe,G.Subramanian,A.Schaffer,D.James, G.Ketawala,N.Venugopalan,S.Xu,S.Corcoran,D.Ferguson,U.Weierstall, J.C.H.Spence,V.Cherezov,P.Fromme,R.F.Fischetti,W.Liu

Key ref:

J.M.Martin-Garcia et al. (2017). Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. IUCrJ, 4, 439-454. PubMed id: 28875031

Date:

20-Feb-17 Release date: 24-May-17

Protein chain

P06873  (PRTK_PARAQ) -  Proteinase K from Parengyodontium album

Seq: 384 a.a.

Struc: 279 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.21.64 - peptidase K.
• Reaction: Hydrolysis of keratin and of other proteins, with subtilisin-like specificity. Hydrolyzes peptides amides.

IUCrJ 4:439-454 (2017)

PubMed id: 28875031

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.

J.M.Martin-Garcia, C.E.Conrad, G.Nelson, N.Stander, N.A.Zatsepin, J.Zook, L.Zhu, J.Geiger, E.Chun, D.Kissick, M.C.Hilgart, C.Ogata, A.Ishchenko, N.Nagaratnam, S.Roy-Chowdhury, J.Coe, G.Subramanian, A.Schaffer, D.James, G.Ketwala, N.Venugopalan, S.Xu, S.Corcoran, D.Ferguson, U.Weierstall, J.C.H.Spence, V.Cherezov, P.Fromme, R.F.Fischetti, W.Liu.

ABSTRACT

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A_2A adenosine receptor (A_2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A_2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A_2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.