2025-09-24 @ CHESS 7b2

The Diffuse Project's first beam time at CHESS.

Goals

• Training of new members
• Commissioning new slits to better define a rectangular top-hat beam
• Collect ambient T data from lysozyme, Mac1 and NrdE crystals

Participants

Steve M, Xiaokun P, Sarah H, Marcus G (Ando lab), John I (CHESS). Steve arrived in the morning to work with John on beamline setup. Marcus joined at 2 pm. Sarah and Xiaokun arrived around 4 pm.

• Xiaokun, Steve, Marcus & Sarah at 7b2 (left-to-right)

• Sarah H mounting a crystal in the 7b2 hutch

• Sarah H collecting lysozyme data

Data

Root directory at CHESS: /nfs/chess/raw/2025-3/id7b2/meisburger/20250924

Beamline setup

parameter	value	notes
X-ray energy	14 keV @ 0.01% bandwidth	Si 111 channel cut mono inserted
Beam size	100 µm x 100 µm, top-hat profile	Defined using slits (newly installed), CRL bypassed (toroidal mirror focusing only)
Flux	3 x 1010 ph/s unattenuated	See station notebook: Steve Meisburger CHESS notebook #3, pg 35
Background reduction	300 µm cleanup aperture moved close to the sample	The in-line camera mirror was removed to make room, so the top-view (90-degree) camera was used for centering
Beamstop	700 µm diameter Mo disk suspended on mylar sheet, semi-transparent	At this energy, the bleedthrough was more intense than usual, and there were some faint diffraction rings visible in the image (mask out?)
Data collection software	"MX Collect" (python) & SPEC	Modified to enable centering with the top view camera. A <prefix>_scan.json file was created for each data collection. Counter values and motor positions were recorded in a <prefix>.spec file. Images in h5 format were generated using Eiger filewriter.
Temperature control	none	The cold stream was removed (ambient T)

• Si 111 channel cut mono

• John installed this new set of JJXray slits downstream of the CRL enclosure

• Top-down view of the sample area (beamstop, goniometer, cleanup aperture, on-axis camera lens)

• Side view of the sample area

• Station computer showing data collection software (MX Collect)

Samples

Name	Sample	Well composition	Drop composition	Notes
Lysozyme	Chicken egg white lysozyme.	NaCl and NaOAc		Practice tray, for calibration and testing purposes only
Mac1	SARS CoV2 NSP3 macrodomain and seed stock from UCSF. 40 mg/mL Mac1 in 150 mM NaCl, 20 mM Tris pH 8, 5% glycerol	34% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5)	2 µl protein solution + 1 ul well solution + 1 ul seeds	Mac1 tray #1. Set up by Katie Lu, 9/19/2025 (see Katie Lu lab notebook pgs. 4-4)
NrdE	Bacillus subtilis class Ib ribonucleotide reductase alpha subunit. 4.5 mg/mL Holo-NrdE protein in 50 mM HEPES pH=7.6, 50 mM NaCl, 5 mM MgCl2, 2 mM TCEP, 1% glycerol, 5 mM ATP, 1 mM ADP, 0.5 mM dGTP	50 mM HEPES (pH=7), 4.5%-6% (w/vol) PEG 3350, 1% tryptone	1:1 protein:well solution (4-5 µL total volume)	Tray set up by Marcus Gamboa on 9/12/2025 (see Marcus Gamboa lab notebook p. 44-50)

• Well A4 of Mac1 tray #1. Well solution: 34% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5). Drop: 2 µl protein solution + 1 ul well solution + 1 ul seeds.

• Well A3 of Bs NrdE Tray #1. Well solultion: 50 mM HEPES (pH=7), 6% (w/vol) PEG 3350, 1% tryptone (total volume of 250 uL) Drop: 2 µl protein solution + 2 ul well solution.

• Well A6 of Bs NrdE Tray #1. Well solution: 50 mM HEPES (pH=7), 4.5% (w/vol) PEG 3350, 1% tryptone (total volume of 250 uL). Drop: 2 µl protein solution + 2 ul well solution.

• Well B4 of Bs NrdE Tray #1. Well solution: 50 mM HEPES (pH=7), 5.5% (w/vol) PEG 3350, 1% tryptone (total volume of 250 uL). Drop: 2 µl protein solution + 2 ul well solution.

• Well C4 of Bs NrdE Tray #1. Well solution: 50 mM HEPES (pH=7), 5.5% (w/vol) PEG 3350, 1% tryptone (total volume of 500 uL). Drop: 2.5 µl protein solution + 2.5 ul well solution.

Data collection

All samples are harvested in a chamber at ~100% relative humidity. Reusable MiTeGen bases are used with MicroRT capillaries cut to length, with 10 µL of well solution in the tip.

Lysozyme

Crystal #1

Steve looped a large lysozyme crystal in a ~400 µm loop.

Subdirectory: lysozyme/calibration_sample

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
lys_cal1_30	0	360	0.1	3600	0.1	880.5	14

Bad dataset. I accidentally left the detector back. Also, 0.1 seconds looks like too much dose, try reducing.

Move to new (undamaged) position. Collect 720 degrees to test for radiation damage in processing:

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
lys_cal1_31	0	720	0.1	7200	0.01	185	14

Should have changed this prefix to lys_cal2, to indicate a new position was used.

Move off the end of the loop for background data collection (translated horizontally along spindle axis)

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
lys_cal_bg_32	0	720	1	720	0.1	185	14

Saved crystal images at two φ angles:

• lys_cal_postcollect_235deg_topview_zoom4_1.png
• lys_cal_postcollect_325deg_topview_zoom4_1.png

Crystal #2

Sarah looped another ~400 µm lysozyme crystal

Subdirectory: lysozyme/calibration_sample

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
lys_cal3_33	0	720	0.1	7200	0.03	185	14
lys_cal3_bg_34	0	720	1	720	0.3	185	14

Saved images:

• lys_cal3_225deg_topview_zoom4_1.png

There has been significant beam drift. Steve tuned mond and mith angles, ICol count rate increased from 8,000 to 16,000 per 0.1 s.

Crystal #3

Third sample: Sarah looped another ~400 µm lysozyme crystal

Subdirectory: lysozyme/calibration_sample

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
lys_cal4_35	0	720	0.1	7200	0.03	185	14
lys_cal4_bg_36	0	720	1	720	0.3	185	14

The background here might not be a good match: although it was positioned off the end of the loop, may not be aligned with the rotation axis (clicked in centering window accidentally)

Saved image:

• lys_cal4_35deg_topview_zoom4_1.png

NrdE

Crystal #4

Xiaokun looped a ~450 µm long BsNrdE crystal from well B4 of Marcus' Bs NrdE Tray #1.

Subdirectory: lysozyme/calibration_sample (forgot to change)

Used vector mode to scan along the length of the crystal (total distance along spindle axis: ∆x).

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	∆x (µm)	E (keV)
lys_cal4_bg_37	0	360	0.1	3600	0.02	225	453.92	14
nrdE_bg_38	0	360	1	360	0.2	225		14

ICol counts ~12,000 per 0.1 s.

Forgot to change prefix (lys_cal4_bg_37 is actually NrdE crystal).

Saved crystal image (same directory):

• nrdE_305deg_postcollection_topview_zoom4_1.png

Crystal #5

Xiaokun looped a ~500 µm BsNrdE crystal from Marcus' same tray, well A3.

Vector scan along the length of the crystal.

Subdirectory: BsNrdE/

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	∆x (µm)	E (keV)
MG_A3_39	115	475	0.1	3600	0.03	250	428.23	14
MG_A3_bg_40	115	475	1	360	0.3	250		14

ICol counts ~14,000 per 0.1 s.

Saved crystal image:

• MG_A3_220deg_postcollection_topview_zoom4_1.png

Crystal #6

Xiaokun looped a ~500 µm BsNrdE crystal from Marcus' tray, well A6

Vector scan along the length of the crystal.

Subdirectory: BsNrdE/

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	∆x (µm)	E (keV)
MG_A6_41	40	475	0.1	4350	0.02	225	340.97	14
MG_A6_bg_42	40	475	1	435	0.2	225		14

ICol counts ~14,000 per 0.1 s.

Saved crystal image:

• MG_A6_75deg_postcollection_topview_zoom4_1.png

Crystal #7

Xiaokun looped a ~400 µm BsNrdE crystal from Macrus' tray, well C4

Vector scan along the length of the crystal.

Subdirectory: BsNrdE/

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	∆x (µm)	E (keV)
MG_C4_43	-285	435	0.1	7200	0.02	225	284.31	14
MG_C4_bg_44	-285	435	1	720	0.2	225		14

ICol counts ~13,000 per 0.1 s.

Saved crystal image:

• MG_C4_220deg_postcollection_topview_zoom4_1.png

Mac1

Crystal #8

10:20 pm. Steve opened well A4 from Katie's macrodomain tray. Looped a ~200 µm Mac1 crystal (200 µm loop, 10 µL of well solution in MicroRT sleeve).

Subdirectory: mac1/mac1_1

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
mac1_1_45	0	720	0.1	7200	0.03	900	14

Oops, forgot to move the detector back. Closed the shutter after a few seconds to avoid damaging the crystal. Repeat the data collection with correct distance:

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
mac1_1_46	0	720	0.1	7200	0.03	185	14
mac1_1_bg_47	0	720	1	720	0.3	185	14

ICol dropped to 3,200 per 0.1 s -- woah, really needs a tuneup.

Saved images at 15, 105, 195 degrees:

• mac1/mac1_1/mac1_1_15deg_postcollection_topview_zoom4_1.png
• mac1/mac1_1/mac1_1_105deg_postcollection_topview_zoom4_1.png
• mac1/mac1_1/mac1_1_195deg_postcollection_topview_zoom4_1.png

Tuned up: ICol 3200 --> 16,000 counts per 0.1 s

For visualization purposes, the second, small oscillation (5 degree) dataset was taken face-on in the same location as the first dataset.

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
mac1_1_48	105	110	0.1	50	1	185	14
mac1_1_bg_49	105	110	0.1	50	1	185	14

Crystal #9

10:51 pm. Steve looped another macrodomain crystal from Well A4 of Katie's tray

Subdirectory: mac1/mac1_2

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
mac1_3_50	0	720	0.1	7200	0.03	185	14
mac1_2_bg_51	0	720	1	720	0.3	185	14
mac1_2_52	110	115	0.1	50	1	185	14
mac1_2_bg_53	110	115	0.1	50	1	185	14

Note, the first prefix contains a typo, it should have been mac1_2 to indicate the second crystal.

ICol counts ~16,000 per 0.1 s.

Forgot to save crystal images (oops)

Crystal #10

11:10 pm. Steve looped another macrodomain crystal from the same well.

Subdirectory: mac1/mac1_3

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
mac1_3_54	0	720	0.1	7200	0.03	185	14
mac1_3_bg_55	0	720	1	720	0.3	185	14
mac1_3_56	20	25	0.1	50	1	185	14
mac1_3_bg_57	20	25	0.1	50	1	185	14

Note: mac1_3_56 went right down a symmetry axis, the diffraction images are really pretty.

ICol ~15,000 counts per 0.1 s.

Saved images:

• mac1/mac1_3/mac1_3_110deg_postcollection_topview_zoom4_1.png
• mac1/mac1_3/mac1_3_20deg_postcollection_topview_zoom4_1.png

Crystal #11

The Mac1 crystals tend to sit flat on the loop in the same way. To obtain more unique views, Steve purposefully bent the loop with tweezers.

11:30 pm. Steve used the bent loop to harvest another macrodomain crystal from the same well in Katie's tray. Note that the coverslip has now been open in the humid chamber for over an hour. There might be some changes to the drop composition (nominally higher water content?). The PEG is also starting to form a sticky skin on the drop.

Subdirectory: mac1/mac1_4

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
mac1_4_58	0	720	0.1	7200	0.03	185	14
mac1_4_bg_59	0	720	1	720	0.3	185	14
mac1_4_60	100	105	0.1	50	1	185	14

Note: there appear to be multiple lattices visible in the mac1_4_60 dataset -- further investigation is needed.

ICol counts: ~13,000 per 0.1 s.

Saved images:

• mac1/mac1_4/mac1_4_10deg_postcollect_topview_zoom4_1.png
• mac1/mac1_4/mac1_4_100deg_postcollect_topview_zoom4_1.png
• mac1/mac1_4/mac1_4_280deg_postcollect_topview_zoom4_1.png

Crystal #12

12:06 am. Steve looped another macrodomain crystal from the same well, again using the bent loop.

Subdirectory: mac1/mac1_5

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	d (mm)	E (keV)
mac1_5_61	0	720	0.1	7200	0.03	185	14
mac1_5_bg_62	0	720	1	720	0.3	185	14
mac1_5_63	100	105	0.1	50	1	185	14
mac1_5_bg_64	100	105	0.1	50	1	185	14

ICol counts: ~12,500 per 0.1 s.

Saved images:

• mac1/mac1_5/mac1_5_195deg_postcollect_topview_zoom4_1.png
• mac1/mac1_5/mac1_5_105deg_postcollect_topview_zoom4_1.png

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